Expression of class 2 mannosidase and class III mannosidase in lower eukaryotic cells

ABSTRACT

A method for producing human-like glycoproteins by expressing a Class 2 α-mannosidase having a substrate specificity for Manα1,3 and Manα1,6 glycosidic linkages in a lower eukaryote is disclosed. Hydrolysis of these linkages on oligosaccharides produces substrates for further N-glycan processing in the secretory pathway.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of U.S. applicationSer. No. 10/371,877, filed on Feb. 20, 2003, which is acontinuation-in-part of U.S. application Ser. No. 09/892,591, filed Jun.27, 2001, which claims the benefit under 35 U.S.C. §119(e) of U.S.Provisional Application No. 60/214,358, filed Jun. 28, 2000, U.S.Provisional Application No. 60/215,638, filed Jun. 30, 2000, and U.S.Provisional Application No. 60/279,997, filed Mar. 30, 2001, each ofwhich is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

[0002] The present invention relates to the field of proteinglycosylation in lower eukaryotes, specifically the introduction of amannosidase enzyme having substrate specificity for hydrolysis ofManα1,3 and/or Manα1,6 glycosidic linkages. The present inventionfurther relates to novel host cells comprising genes encoding amannosidase enzyme and N-glycan or N-glycan-containing intermediatesproduced as a result of the hydrolysis.

BACKGROUND OF THE INVENTION

[0003] Glycosylation Pathways in Humans and Lower Eukaryotes

[0004] After DNA is transcribed and translated into a protein, furtherpost-translational processing involves the attachment of sugar residues,a process known as glycosylation. Different organisms produce differentglycosylation enzymes (glycosyltransferases and glycosidases), and havedifferent substrates (nucleotide sugars) available, so that theglycosylation patterns as well as composition of the individualoligosaccharides, even of the same protein, will be different dependingon the host system in which the particular protein is being expressed.Bacteria typically do not glycosylate proteins, and if so only in a veryunspecific manner (Moens and Vanderleyden, 1997 Arch Microbiol. 168(3):169-175). Lower eukaryotes such as filamentous fungi and yeast addprimarily mannose and mannosylphosphate sugars. The resulting glycan isknown as a “high-mannose” type glycan or a mannan. Plant cells andinsect cells (such as Sf9 cells) glycosylate proteins in yet anotherway. By contrast, in higher eukaryotes such as humans, the nascentoligosaccharide side chain may be trimmed to remove several mannoseresidues and elongated with additional sugar residues that typically arenot found in the N-glycans of lower eukaiyotes. See, e.g., R. K.Bretthauer, et al. Biotechnology and Applied Biochemistry, 1999, 30,193-200; W. Martinet, et al. Biotechnology Letters, 1998, 20, 1171-1177;S. Weikert, et al. Nature Biotechnology, 1999, 17, 1116-1121; M.Malissard, et al. Biochemical and Biophysical Research Communications,2000, 267, 169-173; Jarvis, et al., Current Opinion in Biotechnology,1998, 9:528-533; and M. Takeuchi, 1 Trends in Glycoscience andGlycotechnology, 1997, 9, S29-S35.

[0005] Synthesis of a mammalian-type oligosaccharide structure beginswith a set of sequential reactions in the course of which sugar residuesare added and removed while the protein moves along the secretorypathway in the host organism. The enzymes which reside along theglycosylation pathway of the host organism or cell determine theresulting glycosylation patterns of secreted proteins. Thus, theresulting glycosylation pattern of proteins expressed in lowereukaryotic host cells differs substantially from the glycosylationpattern of proteins expressed in higher eukaryotes such as humans andother mammals (Bretthauer, 1999). The structure of a typical fungalN-glycan is shown in FIG. 1A.

[0006] The early steps of human glycosylation can be divided into atleast two different phases: (i) lipid-linked Glc₃Man₉GlcNAc₂oligosaccharides, are assembled by a sequential set of reactions at themembrane of the endoplasmic reticulum (ER) and (ii) the transfer of thisoligosaccharide from the lipid anchor dolichyl pyrophosphate onto denovo synthesized protein. The site of the specific transfer is definedby an asparagine (Asn) residue in the sequence Asn-Xaa-Ser/Thr where Xaacan be any amino acid except proline (Gavel and von Heijne, 1990 ProteinEng. 3:433-42). Further processing by glucosidases and mannosidasesoccurs in the ER before the nascent glycoprotein is transferred to theearly Golgi apparatus, where additional mannose residues are removed byGolgi specific alpha (α-1,2-) mannosidases. Processing continues as theprotein proceeds through the Golgi. In the medial Golgi, a number ofmodifying enzymes, including N-acetylglucosaminyl Transferases (GnTI,GnTII, GnTIII, GnTIV and GnTV), mannosidase II and fucosyltransferases,add and remove specific sugar residues. Finally, in the trans-Golgi,galactosyltranferases (GalT) and sialyltransferases (ST) produce aglycoprotein structure that is released from the Golgi. It is thisstructure, characterized by bi-, tri- and tetra-antennary structures,containing galactose, fucose, N-acetylglucosamine and a high degree ofterminal sialic acid, that gives glycoproteins their humancharacteristics. The structure of a typical human N-glycan is shown inFIG. 1B.

[0007] In nearly all eukaryotes, glycoproteins are derived from a commonlipid-linked oligosaccharide precursorGlc₃Man₉GlcNAc₂-dolichol-pyrophosphate. Within the endoplasmicreticulum, synthesis and processing of dolichol pyrophosphate boundoligosaccharides are identical between all known eukaryotes. However,further processing of the core oligosaccharide by fungal cells, e.g.,yeast, once it has been transferred to a peptide leaving the ER andentering the Golgi, differs significantly from humans as it moves alongthe secretory pathway and involves the addition of several mannosesugars.

[0008] In yeast, these steps are catalyzed by Golgi residingmannosyl-transferases, like Och1p, Mnt1p and Mnn1p, which sequentiallyadd mannose sugars to the core oligosaccharide. The resulting structureis undesirable for the production of human-like proteins and it is thusdesirable to reduce or eliminate mannosyltransferase activity. Mutantsof S.cerevisiae, deficient in mannosyl-transferase activity (for exampleoch1 or mnn9 mutants) have been shown to be non-lethal and displayreduced mannose content in the oligosaccharide of yeast glycoproteins,thus more closely resembling oligosaccharides of higher eukaryotes.

[0009] Sugar Nucleotide Precursors

[0010] The N-glycans of animal glycoproteins typically includegalactose, fucose, and terminal sialic acid. These sugars are not foundon glycoprofeins produced in yeast and filamentous fungi. In humans, thefull range of nucleotide sugar precursors (e.g. UDP-N-acetylglucosamine,UDP-N-acetylgalactosamine, CMP-N-acetylneuraminic acid, UDP-galactose,GDP-fucose, etc.) are synthesized in the cytosol and transported intothe Golgi, where they are attached to the core oligosaccharide byglycosyltransferases. (Sommers and Hirschberg, 1981 J. Cell Biol. 91(2):A406-A406; Sommers and Hirschberg 1982 J. Biol. Chem. 257(18): 811-817;Perez and Hirschberg 1987 Methods in Enzymology 138: 709-715).

[0011] Glycosyl transfer reactions typically yield a side product whichis a nucleoside diphosphate or monophosphate. While monophosphates canbe directly exported in exchange for nucleoside triphosphate sugars byan antiport mechanism, diphosphonucleosides (e.g. GDP) have to becleaved by phosphatases (e.g. GDPase) to yield nucleoside monophosphatesand inorganic phosphate prior to being exported. This reaction isimportant for efficient glycosylation; for example, GDPase fromSaccharomyces cerevisiae (S.cerevisiae) has been found to be necessaryfor mannosylation. However that GDPase has 90% reduced activity towardUDP (Beminsone et al., 1994 J. Biol. Chem. 269(1):207-211). Lowereukaryotes typically lack UDP-specific diphosphatase activity in theGolgi since they do not utilize UDP-sugar precursors for Golgi-basedglycoprotein synthesis. Schizosaccharomyces pombe, a yeast found to addgalactose residues to cell wall polysaccharides (from UDP-galactose) hasbeen found to have specific UDPase activity, indicating the potentialrequirement for such an enzyme (Beminsone et al. (1994) J. Biol. Chem.269(1):207-211). UDP is known to be a potent inhibitor ofglycosyltransferases and the removal of this glycosylation side productmay be important to prevent glycosyl-transferase inhibition in the lumenof the Golgi (Khatara et al., 1974). See Berninsone, P., et al. 1995. JBiol. Chem. 270(24): 14564-14567; Beaudet, L., et al. 1998 AbcTransporters: Biochemical, Cellular, and Molecular Aspects. 292:397-413.

[0012] Sequential Processing of N-glycans by Compartmentalized EnzymeActivities

[0013] Sugar transferases and glycosidases (e.g., mannosidases) line theinner (luminal) surface of the ER and Golgi apparatus and therebyprovide a “catalytic” surface that allows for the sequential processingof glycoproteins as they proceed through the ER and Golgi network. Themultiple compartments of the cis, medial, and trans Golgi and thetrans-Golgi Network (TGN), provide the different localities in which theordered sequence of glycosylation reactions can take place. As aglycoprotein proceeds from synthesis in the ER to full maturation in thelate Golgi or TGN, it is sequentially exposed to different glycosidases,mannosidases and glycosyltransferases such that a specific carbohydratestructure may be synthesized. Much work has been dedicated to revealingthe exact mechanism by which these enzymes are retained and anchored totheir respective organelle. The evolving picture is complex but evidencesuggests that stem region, membrane spanning region and cytoplasmictail, individually or in concert, direct enzymes to the membrane ofindividual organelles and thereby localize the associated catalyticdomain to that locus (see, e.g., Gleeson, P. A. (1998) Histochem. CellBiol. 109, 517-532).

[0014] In some cases, these specific interactions were found to functionacross species. For example, the membrane spanning domain of α2,6-STfrom rats, an enzyme known to localize in the trans-Golgi of the animal,was shown to also localize a reporter gene (invertase) in the yeastGolgi (Schwientek et al. (1995) J. Biol. Chem. 270(10):5483-9). However,the very same membrane spanning domain as part of a full-length α2,6-STwas retained in the ER and not further transported to the Golgi of yeast(Krezdom et al. (1994) Eur. J. Biochem. 220(3):809-17). A full lengthGalT from humans was not even synthesized in yeast, despite demonstrablyhigh transcription levels. In contrast, the transmembrane region of thesame human GalT fused to an invertase reporter was able to directlocalization to the yeast Golgi, albeit at low production levels.Schwientek and co-workers have shown that fusing 28 amino acids of ayeast mannosyltransferase (MNT1), a region containing a cytoplasmictail, a transmembrane region and eight amino acids of the stem region,to the catalytic domain of human GalT are sufficient for Golgilocalization of an active GalT.

[0015] Other galactosyltransferases appear to rely on interactions withenzymes resident in particular organelles because, after removal oftheir transmembrane region, they are still able to localize properly.

[0016] Improper localization of a glycosylation enzyme may preventproper functioning of the enzyme in the pathway. For example,Aspergillus nidulans, which has numerous α-1,2-mannosidases (Eades andHintz, 2000 Gene 255(1):25-34), does not add GlcNAc to Man₅GlcNAc₂ whentransformed with the rabbit GnTI gene, despite a high overall level ofGnTI activity (Kalsner et al. (1995) Glycoconj. J. 12(3):360-370). GnTI,although actively expressed, may be incorrectly localized such that theenzyme is not in contact with both of its substrates: UDP-GlcNAc and aproductive Man₅GlcNAc₂ substrate (not all Man₅GlcNAc₂ structures areproductive; see below). Alternatively, the host organism may not providean adequate level of UDP-GlcNAc in the Golgi or the enzyme may beproperly localized but nevertheless inactive in its new environment. Inaddition, Man₅GlcNAc₂ structures present in the host cell may differ instructure from Man₅GlcNAc₂ found in mammals. Maras and coworkers foundthat about one third of the N-glycans from cellobiohydrolase I (CBHI)obtained from T.reesei can be trimmed to Man₅GlcNAc₂ by A.saitoi 1,2mannosidase in vitro. Fewer than 1% of those N-glycans, however, couldserve as a productive substrate for GnTI. Maras et al., 1997, Eur. J.Biochem. 249, 701-707. The mere presence of Man₅GlcNAc₂, therefore, doesnot assure that further in vivo processing of Man₅GlcNAc₂ can beachieved. It is formation of a productive, GnTI-reactive Man₅GlcNAc₂structure that is required. Although Man₅GlcNAc₂ could be produced inthe cell (about 27 mol %), only a small fraction could be converted toMan₅GlcNAc₂ (less than about 5%, see Chiba WO 01/14522).

[0017] To date, there is no reliable way of predicting whether aparticular heterologously expressed glycosyltransferase or mannosidasein a lower eukaryote will be (1) sufficiently translated, (2)catalytically active or (3) located to the proper organelle within thesecretory pathway. Because all three of these are necessary to affectglycosylation patterns in lower eukaryotes, a systematic scheme toachieve the desired catalytic function and proper retention of enzymesin the absence of predictive tools, which are currently not available,would be desirable.

[0018] Production of Therapeutic Glycoproteins

[0019] A significant number of proteins isolated from humans or animalsare post-translationally modified, with glycosylation being one of themost significant modifications. An estimated 70% of all therapeuticproteins are glycosylated and thus currently rely on a production system(i.e., host cell) that is able to glycosylate in a manner similar tohumans. Several studies have shown that glycosylation plays an importantrole in determining the (1) immunogenicity, (2) pharmnacokineticproperties, (3) trafficking and (4) efficacy of therapeutic proteins. Itis thus not surprising that substantial efforts by the pharmaceuticalindustry have been directed at developing processes to obtainglycoproteins that are as “humanoid” or “human-like” as possible. Todate, most glycoproteins are made in a mammalian host system. This mayinvolve the genetic engineering of such mammalian cells to enhance thedegree of sialylation (i.e., terminal addition of sialic acid) ofproteins expressed by the cells, which is known to improvepharmacokinetic properties of such proteins. Alternatively, one mayimprove the degree of sialylation by in vitro addition of such sugarsusing known glycosyltransferases and their respective nucleotide sugars(e.g., 2,3-sialyltransferase and CMP-sialic acid).

[0020] While most higher eukaryotes carry out glycosylation reactionsthat are similar to those found in humans, recombinant human proteinsexpressed in the above mentioned host systems invariably differ fromtheir “natural” human counterpart (Raju et al. (2000) Glycobiology10(5): 477-486). Extensive development work has thus been directed atfinding ways to improve the “human character” of proteins made in theseexpression systems. This includes the optimization of fermentationconditions and the genetic modification of protein expression hosts byintroducing genes encoding enzymes involved in the formation ofhuman-like glycoforms. Goochee et al. (1999) Biotechnology9(12):1347-55; Andersen and Goochee (1994) Curr Opin Biotechnol.5(5):546-49; Werner et al. (1998) Arzneimittelforschung. 48(8):870-80;Weikert et al. (1999) Nat Biotechnol. 17(11):1116-21; Yang and Butler(2000) Biotech. Bioeng. 68:370-80. Inherent problems associated with allmammalian expression systems have not been solved.

[0021] Glycoprotein Production Using Eukaryotic Microorganisms

[0022] Although the core oligosaccharide structure transferred to aprotein in the endoplasmic reticulum is basically identical in mammalsand lower eukaryotes, substantial differences have been found in thesubsequent processing reactions which occur in the Golgi apparatus offungi and mammals. In fact, even amongst different lower eukaryotesthere exist a great variety of glycosylation structures. This hashistorically prevented the use of lower eukaryotes as hosts for theproduction of recombinant human glycoproteins despite otherwise notableadvantages over mammalian expression systems.

[0023] Therapeutic glycoproteins produced in a microorganism host suchas yeast utilizing the endogenous host glycosylation pathway differstructurally from those produced in mammalian cells and typically showgreatly reduced therapeutic efficacy. Such glycoproteins are typicallyimmunogenic in humans and show a reduced half-life (and thusbioactivity) in vivo after administration (Takeuchi (1997) Trends inGlycoscience and Glycotechnology 9, S29-S35). Specific receptors inhumans and animals (i.e., macrophage mannose receptors) can recognizeterminal mannose residues and promote the rapid clearance of the foreignglycoprotein from the bloodstream. Additional adverse effects mayinclude changes in protein folding, solubility, susceptibility toproteases, trafficking, transport, compartmentalization, secretion,recognition by other proteins or factors, antigenicity, orallergenicity.

[0024] Yeast and filamentous fungi have both been successfully used forthe production of recombinant proteins, both intracellular and secreted(Cereghino, J. L. and J. M. Cregg 2000 FEMS Microbiology Reviews 24(1):45-66; Harkki, A., et al. 1989 Bio-Technology 7(6): 596; Berka, R. M.,et al. 1992 Abstr.Papers Amer. Chem. Soc. 203: 121-BIOT; Svetina, M., etal. 2000 J. Biotechnol. 76(2-3): 245-251). Various yeasts, such as K.lactis, Pichia pastoris, Pichia methanolica, and Hansenula polymorpha,have played particularly important roles as eukaryotic expressionsystems because they are able to grow to high cell densities and secretelarge quantities of recombinant protein. Likewise, filamentous fungi,such as Aspergillus niger, Fusarium sp, Neurospora crassa and others,have been used to efficiently produce glycoproteins at the industrialscale. However, as noted above, glycoproteins expressed in any of theseeukaryotic microorganisms differ substantially in N-glycan structurefrom those in animals. This has prevented the use of yeast orfilamentous fungi as hosts for the production of many therapeuticglycoproteins.

[0025] Although glycosylation in yeast and fungi is very different thanin humans, some common elements are shared. The first step, the transferof the core oligosaccharide structure to the nascent protein, is highlyconserved in all eukaryotes including yeast, fungi, plants and humans(compare FIGS. 1A and 1B). Subsequent processing of the coreoligosaccharide, however, differs significantly in yeast and involvesthe addition of several mannose sugars. This step is catalyzed bymannosyltransferases residing in the Golgi (e.g. OCH1, MNT1, MNN1,etc.), which sequentially add mannose sugars to the coreoligosaccharide. The resulting structure is undesirable for theproduction of humanoid proteins and it is thus desirable to reduce oreliminate mannosyltransferase activity. Mutants of S.cerevisiaedeficient in mannosyltransferase activity (e.g. och1 or mnn9 mutants)have shown to be non-lethal and display a reduced mannose content in theoligosaccharide of yeast glycoproteins. Other oligosaccharide processingenzymes, such as mannosylphosphate transferases, may also have to beeliminated depending on the host's particular endogenous glycosylationpattern. After reducing undesired endogenous glycosylation reactions,the formation of complex N-glycans has to be engineered into the hostsystem. This requires the stable expression of several enzymes andsugar-nucleotide transporters. Moreover, one has to localize theseenzymes so that a sequential processing of the maturing glycosylationstructure is ensured.

[0026] Several efforts have been made to modify the glycosylationpathways of eukaryotic microorganisms to provide glycoproteins moresuitable for use as mammalian therapeutic agents. For example, severalglycosyltransferases have been separately cloned and expressed in S.cerevisiae (GalT, GnTI), Aspergillus nidulans (GnTI) and other fungi(Yoshida et al. (1999) Glycobiology 9(1):53-8, Kalsner et al. (1995)Glycoconj. J. 12(3):360-370). However, N-glycans resembling those madein human cells were not obtained.

[0027] Yeasts produce a variety of mannosyltransferases (e.g.,1,3-mannosyltransferases such as MNN1 in S. cerevisiae; Graham and Emr,1991 J. Cell. Biol. 114(2):207-218), 1,2-mannosyltransferases (e.g.KTR/KRE family from S.cerevisiae), 1,6-mannosyltransferases (e.g., OCH1from S.cerevisiae), mannosylphosphate transferases and their regulators(e.g., MNN4 and MNN6 from S.cerevisiae) and additional enzymes that areinvolved in endogenous glycosylation reactions. Many of these genes havebeen deleted individually giving rise to viable organisms having alteredglycosylation profiles. Examples are shown in Table 1. TABLE 1 Examplesof yeast strains having altered mannosylation Strain N-glycan (wildtype) Mutation N-glycan (mutant) Reference S. pombe Man_(>9)GlcNAc₂ OCH1Man₈GlcNAc₂ Yoko-o et al., 2001 FEBS Lett. 489(1): 75-80 S. cerevisiaeMan_(>9)GlcNAc₂ OCH1/MNN1 Man₈GlcNAc₂ Nakanishi-Shindo et al,. 1993 J.Biol. Chem. 268(35): 26338-26345 S. cerevisiae Man_(>9)GlcNAc₂OCH1/MNN1/MNN4 Man₈GlcNAc₂ Chiba et al., 1998 J. Biol. Chem. 273,26298-26304 P. pastoris Hyperglycosylated OCH1 (complete Not Welfide,Japanese deletion) hyperglycosylated Application Publication No. 8-336387 P. pastoris Man_(>8)GlcNAc₂ OCH1 (disruption) Man_(>8)GlcNAc₂Contreras et al. WO 02/00856 A2

[0028] Japanese Patent Application Publication No. 8-336387 disclosesthe deletion of an OCH1 homolog in Pichia pastoris. In S.cerevisiae,OCH1 encodes a 1,6-mannosyltransferase, which adds a mannose to theglycan structure Man₈GlcNAc₂ to yield Man₉GlcNAc₂. The Man₉GlcNAc₂structure, which contains three 1,6 mannose residues, is then asubstrate for further 1,2-, 1,6-, and 1,3-mannosyltransferases in vivo,leading to the hypermannosylated glycoproteins that are characteristicfor S.cerevisiae and which typically may have 30-40 mannose residues perN-glycan. Because the Och1p initiates the transfer of 1,6 mannose to theMan₈GlcNAc₂ core, it is often referred to as the “initiating 1,6mannosyltransferase” to distinguish it from other 1,6mannosyltransferases acting later in the Golgi. In an och1 mnn1 mnn4mutant strain of S.cerevisiae, proteins glycosylated with Man₈GlcNAc₂accumulate and hypermannosylation does not occur. However, Man₈GlcNAc₂is not a substrate for mammalian glycosyltransferases, such as humanUDP-GlcNAc transferase I, and accordingly, the use of that mutantstrain, in itself, is not useful for producing mammalian-like proteins,i.e., with complex or hybrid glycosylation patterns.

[0029] One can trim Man₈GlcNAc₂ structures to a Man₅GlcNAc₂ isomer inS.cerevisiae (although high efficiency trimming greater than 50% in vivohas yet to be demonstrated) by engineering a fungal mannosidase from A.saitoi into the endoplasmic reticulum (ER). The shortcomings of thisapproach are two-fold: (1) it is not clear whether the Man₅GlcNAc₂structures formed are in fact forined in vivo (rather than having beensecreted and further modified by mannosidases outside the cell); and (2)it is not clear whether any Man₅GlcNAc₂ structures formed, if in factformed in vivo, are the correct isoform to be a productive substrate forsubsequent N-glycan modification by GlcNAc transferase I (Maras et al.,1997, Eur. J. Biochem. 249, 701-707).

[0030] With the objective of providing a more humanlike glycoproteinderived from a fungal host, U.S. Pat. No. 5,834,251 discloses a methodfor producing a hybrid glycoprotein derived from Trichoderma reseei. Ahybrid N-glycan has only mannose residues on the Manα1-6 arm of the coremannose structure and one or two complex antennae on the Manα1-3 arm.While this structure has utility, the method has the disadvantage thatnumerous enzymatic steps must be performed in vitro, which is costly andtime-consuming. Isolated enzymes are expensive to prepare and needcostly substrates (e.g. UDP-GlcNAc). The method also does not allow forthe production of complex glycans on a desired protein.

[0031] Intracellular Mannosidase Activity Involved in N-glycan Trimming

[0032] Alpha-1,2-mannosidase activity is required for the trimming ofMan₈GlcNAc₂ to form Man₅GlcNAc₂, which is a major intermediate forcomplex N-glycan formnation in mammals. Previous work has shown thattruncated murine, fungal and human α-1,2-mannosidase can be expressed inthe methylotropic yeast P.pastoris and display Man₈GlcNAc₂ toMan₅GlcNAc₂ trimming activity (Lal et al., Glycobiology 1998October;8(10):981-95; Tremblay et al., Glycobiology 1998June;8(6):585-95, Callewaert et al. (2001) FEBS Lett. 503(2-3):173-8).However, to date, no reports exist that show the high level in vivotrimming of Man₈GlcNAc₂ to Man₅GlcNAc₂ on a secreted glycoprotein fromP.pastoris.

[0033] Moreover, the mere presence of an α-1,2-mannosidase in the celldoes not, by itself, ensure proper intracellular trimming of Man₈GlcNAc₂to Man₅GlcNAc₂. (See, e.g., Contreras et al. WO 02/00856 A2, in which anHDEL tagged mannosidase of T. reesei is localized primarily in the ERand co-expressed with an influenza haemagglutinin (HA) reporter proteinon which virtually no Man₅GlcNAc₂ could be detected. See also Chiba etal. (1998) J. Biol. Chem. 273(41): 26298-26304, in which a chimericα-1,2-mannosidase/Och1p transmembrane domain fusion localized in the ER,early Golgi and cytosol of S.cerevisiae, had no mannosidase trimmingactivity). Accordingly, mere localization of a mannosidase in the ER orGolgi is insufficient to ensure activity of the respective enzyme inthat targeted organelle. (See also, Martinet et al. (1998) Biotech.Letters 20(12): 1171-1177, showing that α-1,2-mannosidase from T.reesei, while localizing intracellularly, increased rather thandecreased the extent of mannosylation). To date, there is no report thatdemonstrates the intracellular localization of an active heterologousα-1,2-mannosidase in either yeast or fungi using a transmembranelocalization sequence.

[0034] While it is useful to engineer strains that are able to produceMan₅GlcNAc₂ as the primary N-glycan structure, any attempt to furthermodify these high mannose precursor structures to more closely resemblehuman glycans requires additional in vivo or in vitro steps. Methods tofurther humanize glycans from fungal and yeast sources in vitro aredescribed in U.S. Pat. No. 5,834,251 (supra). If Man₅GlcNAc₂ is to befurther humanized in vivo, one has to ensure that the generatedMan₅GlcNAc₂ structures are, in fact, generated intracellularly and notthe product of mannosidase activity in the medium. Complex N-glycanformation in yeast or fungi will require high levels of Man₅GlcNAc₂ tobe generated within the cell because only intracellular Man₅GlcNAc₂glycans can be further processed to hybrid and complex N-glycans invivo. In addition, one has to demonstrate that the majority ofMan₅GlcNAc₂ structures generated are in fact a substrate for GnTI andthus allow the formnation of hybrid and complex N-glycans.

[0035] Accordingly, the need exists for methods to produce glycoproteinscharacterized by a high intracellular Man₅GlcNAc₂ content which can befurther processed into human-like glycoprotein structures in non-humaneukaryotic host cells, and particularly in yeast and filamentous fungi.

[0036] Class 2 Mannosidases

[0037] A number of class 2 mannosidases of have been purified andcharacterized: mouse mannosidase II, human mannosidase II and Drosophilamannosidase II (FIG. 24 shows a phylogenetic tree of the classes ofmannosidases). It has been found that Class 2 mannosidase enzymes areresponsible for the hydrolysis of α1,3 and α1,6 mannose glycosidiclinkages on N-linked oligosaccharides generally localized in the Golgi.At least five types of Class 2 mannosidases have been identified,namely: (1) Golgi α-mannosidase II; (2) Golgi α-mannosidase IIx; (3)lysosomal α-mannosidase; (4) cytosolic α-mannosidase; and (5) an enzymecharacterized from mouse and pig sperm or epididymal tissues. Moremen K.W., Biochimica Biophysica Acta 1573 (2002) 225-235.

[0038] Human congenital dyserythropoietic anemia type II has beenassociated with the lack of functional α-mannosidase II gene asexhibited in mice. Chui et al. Cell 1997 July 11 ;90(1):157-67. Thisgenetic defect is referred to as HEMPAS (hereditary erythroblasticmultinuclearity with positive acidified serum lysis test), and furtherresearch is underway to study the role of α-mannosidase II. For example,a mutation of a single gene encoding α-mannosidase II has been shown toresult in a systemic autoimmune disease similar to human systemic lupuserythematosis. Chui et al., Proc. Natl. Acad. Sci. USA 200198:1142-1147.

[0039] The importance of the enzymatic activity in glycoproteinformation has been well-established; however, efficient expression ofsuch activity for the production of therapeutic glycoproteins has notbeen accomplished in lower eukaryotic cells.

[0040] (1) Golgi α-mannosidase II

[0041] The Golgi α-mannosidase II (EC. 3.2.1.114) is a Type IItransmembrane protein, approximately 125 kDa in size, composed of ashort N-terminal cytoplasmic tail, a single-span transmembrane domainconnected by a stalk segment to a large luminal C-terminal catalyticportion. Moremen and Touster, J. Biol. Chem., 260, 6654-6662; Moremenand Touster, J. Biol. Chem., 261, 10945-10951. The function of themannosidase II is essential in the processing of N-glycans in thesecretory pathway. In mammalian cells, it has been established that thisparticular enzyme hydrolyzes the Manα1,3 and Manα1,6 glycosidic linkageson the substrate GlcNAcMan₅GlcNAc₂. Subsequent N-glycan processing iscatalyzed by other glycosylation enzymes (e.g.N-acetylglucosaminyltransferases, galactosyltransferases,sialyltransferases) to produce the desired glycoforms with theirsubstrates (UDP-GlcNAc, UDP-GalNAc, CMP-Sialic acid) and theirrespective transporters. See, e.g., WO 02/00879, which is incorporatedby reference herein in its entirity.

[0042] A partial clone encoding the Golgi α-mannosidase II has beenisolated from a rat liver λgt11 cDNA library. Moremen, K W. Proc. Natl.Acad. Sci. USA 1989 July;86(14):5276-80. The mouse Golgi α-mannosidaseII and the human α-mannosidase II have also been characterized andexpressed in COS cells. Moremen and Robbins, J. Cell. Biol. 1991December;115(6):1521-34. Research conducted on Golgi α-mannosidase IIenzyme shows that there is considerable similarity within the C-terminaldomain of this class of enzyme. In addition, substrate specificitystudies show that the hydrolysis of the α1,3 and/or α1,6 glycosidiclinkages by the Golgi α-mannosidase II enzyme requires a terminal GlcNAcon the oligosaccharide substrate.

[0043] The Drosophila melangaster Golgi α-mannosidase II has beenisolated using the murine Golgi α-mannosidase II cDNA and is shown tohave considerable similarity to regions from other α-mannosidases.Foster et al. Gene 154 (1995) 183-186. Previous work has shown that theDrosophila and mouse cDNA sequences translate amino acid sequences of41% identity and 61% similarity. Expression of the Drosophila Golgiα-mannosidase II sequence in CHOP cells (CHO cells stably expressingpolyoma large T-antigen) was shown to be active and was also shown tolocalize mainly in the Golgi apparatus. Rabouille et al. J. Cell. Sci.1999 October;112 (Pt 19):3319-30.

[0044] (2) Golgi α-mannosidase IIx

[0045] The gene encoding the human α-mannosidase IIx (α-mannosidase IIisotype) has been characterized. Ogawa et al. Eur. J. Biochem. 242,446-453 (1996). Overexpression of the α-mannosidase IIx enzyme leads tothe conversion of Man₆GlcNAc₂ to Man₄GlcNAc₂ in CHO cells. Oh-eda et al.Eur. J. Biochem. 268, 1280-1288 (2001). The two types of mannosidases(II and IIx) are closely related to the processing of N-glycans in theGolgi. This Golgi α-mannosidase IIx has 66% identity to α-mannosidase IIand has similar catalytic activity of hydrolyzing the Manα1,6 andManα1,3 of the Man₆GlcNAc₂ oligosaccharide. More recent studies revealedan obvious phenotype of infertility in α-mannosidase IIx-deficient malemice. Biochim Biophys Acta. 2002 Dec. 19;1573(3):382-7. One study foundthat α-mannosidase IIx-deficient mouse testis showed reduced levels ofGlcNAc-terminated complex type N-glycans.

[0046] (3) Lysosomal α-mannosidase

[0047] Another type of Class 2 mannosidase is found in the lysosome ofeukaryotic cells and is involved in glycoprotein catabolism (breakdown).Unlike the Golgi mannosidase II enzyme, which has a neutral pH optimum,the lysosomal mannosidase II has a low pH optimum (pH 4.5), has broadnatural substrate specificity, is active toward the synthetic substratep-nitrophenyl-α-mannosidase and is sensitive to inhibition byswainsonine. Daniel et al., (1994) Glycobiology 4, 551-566; Moremen etal., (1 994) Glycobiology 4, 113-125. Structurally, the lysosomalα-mannosidase has an N-terminal signal sequence in place of thecytoplasmic tail, transmembrane domain, and stem region of the Golgienzyme. Moremen, K. W., Biochimica Biophysica Acta 1573 (2002) 225-235.The human lysosomal α-mannosidase (EC 3.2.1.24) has been cloned andexpressed in Pichia pastoris. Liao et al., J Biol Chem 1996 Nov.8;271(45):28348-58. Based on regions of amino acid sequence conservationbetween the lysosomal α-mannosidase from Dictyostelium discoideum andthe murine Golgi α-mannosidase II (a glycoprotein that processesα1,3/1,6-mannosidase activity) a cDNA encoding the murine lysosomalα-mannosidase was cloned. Merkle et al., Biochim Biophys Acta 1997 Aug.29; 1336(2):132-46. A deficiency in the lysosomal α-mannosidase resultsin a human genetic disease termed α-mannosidosis.

[0048] (4) Cytosolic α-mannosidase

[0049] The cytosolic α-mannosidase II is less-similar to the other Class2 mannosidases and appears to prefer Co²⁺ over Zn²⁺ for catalyticactivity. Moremen, K. W., Biochimica Biophysica Acta 1573 (2002)225-235. Like the lysosomal α-mannosidase II, it is involved in thecatabolism of glycoproteins. The cytosolic α-mannosidase II catabolizesthe improperly folded glycoproteins in the lumen of the ER that havebeen retro-translocated into the cytoplasm for proteolytic disposal.Duvet et al., Biochem. J. 335 (1998) 389-396; Grard et al., Biochem. J.316 (1996) 787-792. Structurally, this enzyme has no cleavable signalsequence or transmembrane domain.

[0050] Additional mannosidases that exhibit characteristics of Class 2mannosidases have been described but have yet to be cloned for directcomparision by sequence alignment. Moremen, K. W., Biochimica BiophysicaActa 1573 (2002) 225-235.

[0051] Class III Mannosidases

[0052] Class III mannosidases, which are also involved in trimming ofthe Manα1,3 and Manα1,6 glycosidic linkages of an oligosaccharide, e.g.converting Man₅GlcNAc₂ to Man₃GlcNAc₂, have been recently cloned andidentified. To date only two members of this class of proteins areknown. The first member identified was from an anemic mouse that wasdeficient in the classic Golgi mannosidase II activity but possessed analternative mechanism for converting Man₅GlcNAc₂ directly toMan₃GlcNAc₂, which was independent of the presence of GlcNAc on the coremannose-1,3 branch (D. Chui, et al. Cell 1997 90:157-167). This classIII mannosidase has yet to be cloned but a protein with similar activityhas been cloned from Sf9 cells (Z. Kawar, et al. J. Biol. Chem. 2001276(19):16335-16340).

[0053] The only member of the class III mannosidases to be cloned andcharacterized originates from lepidopteran insect cell line Sf9 (D.Jarvis, et al. Glycobiology 1997 7:113-127). This Sf9 Golgi mannosidaseIII converts Man₅GlcNAc₂ to Man₃GlcNAc₂, and, unlike the Golgimannosidase II, does not process GlcNAcMan₅GlcNAc₂. A unique feature ofthis class of mannosidases is that, in addition to possessingManα1,3/1,6 activity, they also possess α-1,2 mannosidase activity likea class I Golgi mannosidase. Furthermore, like the Golgi mannosidase Ienzymes, this Sf9 mannosidase III trims Man₈GlcNAc₂ more efficientlythan Man₉GlcNAc₂.

[0054] Given the utility of the mannosidase enzyme activities inprocessing N-glycans, it would be desirable to have a method forproducing human-like glycoproteins in lower eukaroytic host cellscomprising the step of expressing a catalytically active α-mannosidaseII having substrate specificity for Manα1,3 and Manα1,6 on anoligosaccharide.

SUMMARY OF THE INVENTION

[0055] The invention provides a method for producing a human-likeglycoprotein in a lower eukaryotic host cell comprising the step ofexpressing a catalytically active fragment of a Class 2 or a Class IIImannosidase enzyme.

[0056] One embodiment of the invention provides a method for producing ahuman-like glycoprotein in a lower eukaryotic host cell comprising thestep of expressing in the cell a mannosidase enzymatic activity that iscapable of hydrolyzing an oligosaccharide substrate comprising either orboth a Manα1,3 and Manα1,6 glycosidic linkage to the extent that atleast 10% of the Manα1,3 and/or Manα1,6 linkages of the substrate arehydrolyzed in vivo.

[0057] Another embodiment of the invention provides a method forproducing a desired N-glycan in a lower eukaryotic host cell comprisingthe step of expressing in the cell a mannosidase enzymatic activity thatis capable of hydrolyzing in vivo an oligosaccharide substratecomprising either or both a Manα1,3 and Manα1,6 glycosidic linkagewherein the desired N-glycan is produced within the host cell at a yieldof at least 10 mole percent.

[0058] Preferably, the desired N-glycan produced is selected from thegroup consisting of Man₃GlcNAc₂, GlcNAcMan₃GlcNAc₂ and Man₄GlcNAc₂. Inanother preferred embodiment, the desired N-glycan is characterized ashaving at least the oligosaccharide branch Manα1,3 (Manα1,6)Manβ1,4-GlcNAc β1,4-GlcNAc β1-Asn. The glycoprotein is preferablyisolated from the host cell. In yet another preferred embodiment, themannosidase enzymatic activity is capable of hydrolyzing in vivo bothManα1,3 and Manα1,6 linkages of an oligosaccharide substrate comprisinga Manα1,3 and Manα1,6 glycosidic linkage.

[0059] In another preferred embodiment, the oligosaccharide substrate ischaracterized as Manα1,3 (Manα1,6 Manα1,6) Manβ1,4-GlcNAcβ1,4-GlcNAc-Asn; Manα1,3 (Manα1,3 Manα1,6) Manβ1,4-GlcNAcβ1,4-GlcNAc-Asn; GlcNAcβ1,2 Manα1,3 (Manα1,6 Manα1,6) Manβ1,4-GlcNAcβ1,4-GlcNAc-Asn; GlcNAcβ1,2 Manα1,3 (Manα1,3 Manα1,6) Manβ1,4-GlcNAcβ1,4-GlcNAc-Asn; Manα1,3 (Manα1,3 Manα1,6 Manα1,6) Manβ1,4-GlcNAcβ1,4-GlcNAc-Asn; GlcNAcβ1,2 Manα1,3 (Manα1,3 Manα1,6 Manα1,6)Manβ1,4-GlcNAc β1,4-GlcNAc-Asn; Manα1,2 Manα1,3 (Manα1,3 Manα1,6Manα1,6) Manβ1,4-GlcNAc β1,4-GlcNAc-Asn; Manα1,2 Manα1,3 (Manα1,3Manα1,6) Manβ1,4-GlcNAc β1,4-GlcNAc-Asn; Manα1,2 Manα1,3 (Manα1,6Manα1,6) Manβ1,4-GlcNAc β1,4-GlcNAc-Asn or high mannan.

[0060] In a preferred embodiment, the mannosidase activity ischaracterized as a Class 2 mannosidase activity. In a more preferredembodiment, the Class 2 mannosidase activity has a substrate specificityfor GlcNAcβ1,2 Manα1,3 (Manα1,6 Manα1,6) Manβ1,4-GlcNAc β1,4-GlcNAc-Asn;GlcNAcβ1,2 Manα1,3 (Manα1,3 Manα1,6) Manβ1,4-GlcNAc β1,4-GlcNAc-Asn; orGlcNAcβ1,2 Manα1,3 (Manα1,3 Manα1,6 Manα1,6) Manβ1,4-GlcNAcβ1,4-GlcNAc-Asn. In an even more preferred embodiment, the Class 2mannosidase activity is one which is normally found in the Golgiapparatus of a higher eukaryotic host cell.

[0061] In another preferred embodiment, the mannosidase activity ischaracterized as a Class IIx mannosidase activity. In a more preferredembodiment, the Class IIx mannosidase activity has a substratespecificity for Manα1,3 (Manα1,6 Manα1,6) Manβ1,4-GlcNAcβ1,4-GlcNAc-Asn; Manα1,3 (Manα1,3 Manα1,6) Manβ1,4-GlcNAcβ1,4-GlcNAc-Asn; or Manα1,2 Manα1,3 (Manα1,3 Manα1,6 Manα1,6)Manβ1,4-GlcNAc β1,4-GlcNAc-Asn.

[0062] In yet another preferred embodiment, the mannosidase activity ischaracterized as a Class III mannosidase activity. In a more preferredembodiment, the Class III mannosidase activity has a substratespecificity for (Manα1,6 Manα1,6) Manβ1,4-GlcNAc β1,4-GlcNAc-Asn;(Manα1,3 Manα1,6) Manβ1,4-GlcNAc β1,4-GlcNAc-Asn; or high mannans.

[0063] In any one of the above embodiments, the mannosidase activity ispreferably overexpressed. In another preferred embodiment, themannosidase is further capable of hydrolyzing a Manα1,2 linkage. Themannosidase activities of the invention preferably have a pH optimum offrom about 5.0 to about 8.0.

[0064] In another embodiment the mannosidase activity is localizedwithin the secretory pathway of the host cell. Preferably, themannosidase activity is expressed from a polypeptide localized within atleast one of the ER, Golgi apparatus or the trans golgi network of thehost cell.

[0065] In one preferred embodiment, the mannosidase activity isexpressed from a nucleic acid encoding a polypeptide comprising amannosidase catalytic domain fused to a cellular targeting signalpeptide. In a more preferred embodiment, the mannosidase activity isexpressed from a nucleic acid comprising sequences that encode amannosidase catalytic domain native to the host cell. In another morepreferred embodiment, the mannosidase activity is expressed from anucleic acid comprising sequences that encode a mannosidase catalyticdomain heterologous to the host cell.

[0066] In another preferred embodiment, the mannosidase enzymaticactivity is selected from the group consisting of βArabidopsis thalianaMannosidase II, C. elegans Mannosidase II, Ciona intestinalismannosidase II, Drosophila mannosidase II, Human mannosidase II, Mousemannosidase II, Rat mannosidase II, Human mannosidase IIx, Insect cellmannosidase III, Human lysosomal mannosidase II and Human cytoplasmicmannosidase II.

[0067] In another preferred embodiment, the polypeptide is expressedfrom a nucleic acid comprising sequences that encode a target peptidenative to the host cell.

[0068] In another preferred embodiment, the polypeptide is expressedfrom a nucleic acid comprising sequences that encode a target peptideheterologous to the mannosidase catalytic domain.

[0069] In a preferredeembodiment, the host cell is selected from thegroup consisting of Pichia pastoris, Pichia finlandica, Pichiatrehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia opuntiae,Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichiapijperi, Pichia stiptis, Pichia methanolica, Pichia sp., Saccharomycescerevisiae, Saccharomyces sp., Hansenula polymorpha, Kluyveromyces sp.,Kluyveromyces lactis, Candida albicans, Aspergillus nidulans,Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporiumlucknowense, Fusarium sp., Fusarium gramineum, Fusarium venenatum andNeurospora crassa. In a more preferred embodiment, the host cell isPichia pastoris.

[0070] The invention further provides glycoproteins and N-glycansproduced by 15, one of the above methods. In a preferred embodiment, theglycoprotein is a therapeutic protein. In a more preferred embodiment,the therapeutic protein is selected from the group consisting oferythropoietin, cytokines, coagulation factors, soluble IgE receptorα-chain, IgG, IgG fragments, IgM, interleukins, urokinase, chymase, ureatrypsin inhibitor, IGF-binding protein, epidermal growth factor, growthhormone-releasing factor, annexin V fusion protein, angiostatin,vascular endothelial growth factor-2, myeloid progenitor inhibitoryfactor-1, osteoprotegerin, (α-1-antitrypsin and α-feto protein.

[0071] The invention further provides a nucleic acid library comprisingat least two different genetic constructs, wherein at least one geneticconstruct comprises a nucleic acid fragment encoding a mannosidase class2, IIx or III catalytic domain ligated in-frame with a nucleic acidfragment encoding a cellular targeting signal peptide which it is notnormally associated with.

[0072] In a preferred embodiment, the mannosidase catalytic domain isselected from the group consisting of: Arabidopsis thaliana MannosidaseII, C. elegans Mannosidase II, Ciona intestinalis mannosidase II,Drosophila mannosidase II, Human mannosidase II, Mouse mannosidase II,Rat mannosidase II, Human mannosidase IIx, Insect cell mannosidase III,Human lysosomal mannosidase II and Human cytoplasmic mannosidase II.

[0073] In another preferred embodiment, the nucleic acid fragmentencoding a cellular targeting peptide is selected from the groupconsisting of: Saccharomyces GLS1, Saccharomyces MNS1, SaccharoniycesSEC12, Pichia SEC, Pichia OCH1, Saccharomyces MNN9, Saccharomyces VAN1,Saccharomyces ANP1, Saccharomyces HOC1, Saccharomyces MNN10,Saccharomyces MNN11, Saccharomyces MNT1, Pichia D2, Pichia D9, PichiaJ3, Saccharomyces KTR1, Saccharomyces KTR2, Kluyveromyces GnTI,Saccharomyces MNN2, Saccharomyces MNN5, Saccharomyces YUR1,Saccharonyces MNN1 and Saccharomyces MNN6.

[0074] Another embodiment of the invention provides a vector comprisinga fusion construct derived from any one of the above libraries linked toan expression control sequence, wherein said cellular targeting signalpeptide is targeted to at least one of the ER, Golgi or trans-Golginetwork. In a more preferred embodiment, the expression control sequenceis inducible or constitutive. In an even more preferred embodiment, thevector, upon expression in a host cell, encodes a mannosidase activityinvolved in producing GlcNAcMan₃GlcNAc₂ Man₃GlcNAc₂ or Man₄GlcNAc₂ invivo.

[0075] Another embodiment of the invention provides a host cellcomprising at least one of the above vectors. In more preferredembodiments, the vector is selected from the group of vectors designatedpKD53, pKD1, pKD5, pKD6 and pKD16.

[0076] Another embodiment of the invention provides a chimericpolypeptide comprising a mannosidase catalytic domain fused in-frame toa targeting signal peptide and, upon expression in a lower eukaryotichost cell, capable of hydrolyzing in vivo an oligosaccharide substratecomprising either or both a Manα1,3 and Manα1,6 glycosidic linkage tothe extent that at least 10% of the Manα1,3 and/or Manα1,6 linkages ofthe substrate are hydrolyzed in vivo.

[0077] Another embodiment of the invention provides a chimericpolypeptide comprising a mannosidase catalytic domain fused in-frame toa targeting signal peptide and, upon expression in a lower eukaryotichost cell, capable of hydrolyzing in vivo an oligosaccharide substratecomprising a Manα1,3, Manα1,6, or Manα1,2 glycosidic linkage to theextent that a detectable moiety of the Manα1,3, Manα1,6 or Manα1,2linkage of the substrate is hydrolyzed in vivo.

[0078] Another embodiment of the invention provides a nucleic acidencoding the above chimeric polypeptide or a host cell comprising theabove chimeric polypeptide.

[0079] Another embodiment of the invention provides a host cellcomprising the above nucleic acid.

[0080] Another embodiment of the invention provides a glycoproteinproduced in the above host cell. In a more preferred embodiment, anN-glycan produced in the host cell is provided. More preferably, theglycoprotein is characterized as uniform.

[0081] Another embodiment of the invention provides an isolatedpolynucleotide comprising or consisting of a nucleic acid sequenceselected from the group consisting of the conserved regions SEQ ID NO:5-SEQ ID NO: 15.

BRIEF DESCRIPTION OF THE DRAWINGS

[0082]FIG. 1A is a schematic diagram of a typical fungal N-glycosylationpathway.

[0083]FIG. 1B is a schematic diagram of a typical human N-glycosylationpathway.

[0084]FIG. 2 depicts construction of a combinatorial DNA library offusion constructs. FIG. 2A diagrams the insertion of a targeting peptidefragment into pCR2.1-TOPO (Invitrogen, Carlsbad, Calif.). FIG. 2B showsthe generated targeting peptide sub-library having restriction sitesNotI-AscI. FIG. 2C diagrams the insertion of a catalytic domain regioninto pJN347, a modified pUC19 vector. FIG. 2D shows the generatedcatalytic domain sub-library having restriction sites NotI, AscI andPacI. FIG. 2E depicts one particular fusion construct generated from thetargeting peptide sub-library and the catalytic domain sub-library.

[0085]FIG. 3 illustrates the M.musculus α-1,2-mannosidase IA openreading frame nucleic acid sequence (SEQ ID NO: 1) and encodedpolypeptide sequence (SEQ ID NO: 2). The sequences of the PCR primersused to generate N-terminal truncations are underlined.

[0086]FIGS. 4A-4F illustrate engineering of vectors with multipleauxotrophic markers and genetic integration of target proteins in the P.pastoris OCH1 locus.

[0087]FIGS. 5A-5E show MALDI-TOF analysis demonstrating production ofkringle 3 domain of human plasminogen (K3) glycoproteins havingMan₅GlcNAc₂ as the predominant N-glycan structure in P. pastoris. FIG.5A depicts the standard Man₅GlcNAc₂ [a] glycan (Glyko, Novato, Calif.)and Man₅GlcNAc₂ +Na+[b]. FIG. 5B shows PNGase—released glycans from K3wild type. The N-glycans shown are as follows: MangGlcNAc₂ [d];Man₁₀GlcNAc₂ [e]; Man₁₁GlcNAc₂ [f]; Man₁₂GlcNAc₂ [g]. FIG. 5C depictsthe och1 deletion resulting in the production of Man₈GlcNAc₂ [c] as thepredominant N-glycan. FIGS. 5D and 5E show the production of Man₅GlcNAc₂[b] after in vivo trimming of Man₈GlcNAc₂ with a chimericα-1,2-mannosidase. The predominant N-glycan is indicated by a peak witha mass (m/z) of 1253 consistent with its identification as Man₅GlcNAc₂[b].

[0088]FIGS. 6A-6F show MALDI-TOF analysis demonstrating production ofIFN-β glycoproteins having Man₅GlcNAc₂ as the predominant N-glycanstructure in P. pastoris. FIG. 6A shows the standard Man₅GlcNAc₂ [a] andMan₅GlcNAc₂ +Na+[b] as the standard (Glyko, Novato, Calif.). FIG. 6Bshows PNGase-released glycans from IFN-β wildtype. FIG. 6C depicts theoch1 knock-out producing Man₈GlcNAc₂ [c]; MangGlcNAc₂ [d]; Man₁₀GlcNAc₂[e]; Man₁₁GlcNAc₂ [f]; Man₁₂GlcNAc₂ [g]; and no production ofMan₅GlcNAc₂ [b]. FIG. 6D shows relatively small amount of Man₅GlcNAc₂[b] among other intermediate N-glycans Man₈GlcNAc₂ [c] to Man₁₂GlcNAc₂[g]. FIG. 6E shows a significant amount of Man₅GlcNAc₂ [b] relative tothe other glycans Man₈GlcNAc₂ [c] and Man₉GlcNAc₂ [d] produced by pGC5(Saccharomyces MNS1(m)/mouse mannosidase IB Δ99). FIG. 6F showspredominant production of Man₅GlcNAc₂ [b] on the secreted glycoproteinIFN-β by pFB8 (Saccharomyces SEC 12 (m)/mouse mannosidase IA Δ187). TheN-glycan is indicated by a peak with a mass (m/z) of 1254 consistentwith its identification as Man₅GlcNAc₂ [b].

[0089]FIG. 7 shows a high performance liquid chromatogram for: (A)Man₉GlcNAc₂ standard labeled with 2-AB (negative control); (B)supernatant of growth medium from P.pastoris, Δ och1 transformed withpFB8 mannosidase, which demonstrates a lack of extracellular mannosidaseactivity in the supernatant; and (C) Man₉GlcNAc₂ standard labeled with2-AB after exposure to T.reesei mannosidase (positive control).

[0090]FIG. 8 shows a high performance liquid chromatogram for: (A)Man₉GlcNAc₂ standard labeled with 2-AB (negative control); (B)supernatant of growth medium from P.pastoris, Δ och1 transformed withpGC5 mannosidase, which demonstrates a lack of extracellular mannosidaseactivity in the supernatant; and (C) Man₉GlcNAc₂ standard labeled with2-AB after exposure to T.reesei mannosidase (positive control).

[0091]FIG. 9 shows a high performance liquid chromatogram for: (A)Man₉GlcNAc₂ standard labeled with 2-AB (negative control); (B)supernatant of growth medium from P.pastoris, Δ och1 transformed withpBC18-5 mannosidase, which demonstrates lack of extracellularmannosidase activity in the supernatant; and (C) supernatant of mediumP.pastoris, Δ och1 transformed with pDD28-3, which demonstrates activityin the supernatant (positive control).

[0092]FIG. 10A-10B demonstrate the activity of an UDP-GlcNAc transporterin the production of GlcNAcMan₅GlcNAc₂ in P. pastoris. FIG. 10A depictsa P.pastoris strain (YSH-3) transformed with a human GnTI but withoutthe UDP-GlcNAc transporter resulting in some production ofGlcNAcMan₅GlcNAc₂ [b] but a predominant production of Man₅GlcNAc₂ [a].FIG. 10B depicts the addition of UDP-GlcNAc transporter from K.lactis ina strain (PBP-3) transformed with the human GnTI, which resulted in thepredominant production of GlcNAcMan₅GlcNAc₂ [b]. The single prominentpeak of mass (m/z) at 1457 is consistent with its identification asGlcNAcMan₅GlcNAc₂ [b] as shown in FIG. 10B.

[0093]FIG. 11 shows a pH optimum of a heterologous mannosidase enzymeencoded by pBB27-2 (Saccharomyces MNN 10 (s)/C. elegans mannosidase IBΔ31) expressed in P.pastoris.

[0094]FIGS. 12A-12C show MALDI-TOF-MS analyses of N-glycans releasedfrom a cell free extract of K.lactis. FIG. 12A shows the N-glycansreleased from wild-type cells, which includes high-mannose typeN-glycans. FIG. 12B shows the N-glycans released from och1 mnn1 deletedcells, revealing a distinct peak of mass (m/z) at 1908 consistent withits identification as Man₉GlcNAc₂ [d]. FIG. 12C shows the N-glycansreleased from och1 mnn1 deleted cells after in vitro α-1,2-mannosidasedigest corresponding to a peak consistent with Man₅GlcNAc₂.

[0095]FIG. 13 shows a MALDI-TOF-MS analysis of N-glycans isolated from akringle 3 glycoprotein produced in a P.pastoris YSH-1 (och1 deletionmutant transformed with α-mannosidase and GnT I) showing a predominantpeak at 1465 m/z corresponding to the mass of GlcNAcMan₅GlcNAc₂ [d].

[0096]FIG. 14 shows a MALDI-TOF-MS analysis of N-glycans isolated from akringle 3 glycoprotein produced in a P.pastoris YSH-1 transformed withD. melanogaster mannosidase II Δ74/S. cerevisiae MNN2(s) showing apredominant peak at 1140 m/z corresponding to the mass ofGlcNAcMan₃GlcNAc₂ [b] and other peaks corresponding to GlcNAcMan₄GlcNAc₂[c] at 1303 mn/z and GlcNAcMan₅GlcNAc₂ [d] at 1465 m/z. This strain wasdesignated YSH-37.

[0097]FIG. 15 shows a MALDI-TOF-MS analysis of N-glycans isolated from akringle 3 glycoprotein produced in a P.pastoris YSH-37 transformed withrat GnT II/MNN2 (s) leader showing a predominant peak at 1356 m/zcorresponding to the mass of GlcNAc₂Man₃GlcNAc₂ [x]. This strain wasdesignated YSH-44.

[0098]FIG. 16 shows a MALDI-TOF-MS analysis of N-glycans isolated from akringle 3 glycoprotein produced in a P.pastoris YSH-44(GlcNAc₂Man₃GlcNAc₂ [b] produced as shown in FIG. 15) showing apredominant peak at 933 m/z corresponding to the mass of Man₃GlcNAc₂ [a]after β-N-acetylhexosaminidase digest.

[0099]FIG. 17 shows a MALDI-TOF-MS analysis of N-glycans isolated from akringle 3 glycoprotein produced in a P.pastoris YSH-44(GlcNAc₂Man₃GlcNAc₂ [b] produced as shown in FIG. 15) showing apredominant peak at 1665 m/z corresponding to the mass ofGal₂GlcNAc₂Man₃GlcNAc₂ after addition of β1,4-galactosyltransferase invitro.

[0100]FIG. 18 shows a MALDI-TOF-MS analysis of N-glycans isolated from akringle 3 glycoprotein produced in a P.pastoris YSH-1 transformed withD. melanogaster mannosidase IIΔ74/S. cerevisiae MNN9(m) showing apredominant peak at 1464 m/z corresponding to the mass of Man₅GlcNAc₂[d].

[0101]FIG. 19 shows a MALDI-TOF-MS analysis of N-glycans isolated from akringle 3 glycoprotein produced in a P.pastoris YSH-1 transformed withD. melanogaster mannosidase IIΔ74/S. cerevisiae MNS1(1) showing apredominant peak at 1464 m/z corresponding to the mass of Man₅GlcNAc₂[d] and other peaks corresponding to GlcNAcMan₃GlcNAc₂ [b] at 1139 m/zand GlcNAcMan₄GlcNAc₂ [c] at 1302 m/z.

[0102]FIG. 20 shows a MALDI-TOF-MS analysis of N-glycans isolated from akringle 3 glycoprotein produced in a P.pastoris YSH-1 transformed withD. melanogaster mannosidase IIΔ74/S. cerevisiae GLS1(s) showing apredominant peak at 1139 m/z corresponding to the mass ofGlcNAcMan₃GlcNAc₂ [b]. This strain was designated YSH-27.

[0103]FIG. 21 shows a MALDI-TOF-MS analysis of N-glycans isolated from akringle 3 glycoprotein produced in a P.pastoris YSH-1 transfonred withD. melanogaster mannosidase IIΔ74/S. cerevisiae MNS1(m) showing apredominant peak at 1140 m/z corresponding to the mass ofGlcNAcMan₃GlcNAc₂ [b] and other peaks corresponding to GlcNAcMan₄GlcNAc₂[c] at 1302 m/z and GlcNAcMan₅GlcNAc₂ [d] at 1464 m/z. This strain wasdesignated YSH-74.

[0104]FIG. 22 shows a MALDI-TOF-MS analysis of N-glycans isolated from akringle 3 glycoprotein produced in a P.pastoris YSH-74 digested with aT. reesei/A. saitoi α-1,2 mannosidase showing a predominant peak at 1141m/z corresponding to the mass of GlcNAcMan₃GlcNAc₂ [b].

[0105]FIG. 23 shows a BLAST Sequence Comparision of known andhypothetical mannosidase II, mannosidase IIx and Class III mannosidases.

[0106]FIG. 24 shows a phylogenetic tree of the classes of mannosidase.

[0107]FIG. 25 shows an Arabidopsis thaliana Mannosidase II(NM_(—)121499) Sequence.

[0108]FIG. 26 shows a C. elegans Mannosidase II (NM_(—)073594) Sequence.

[0109]FIG. 27 shows a Ciona intestinalis mannosidase II (AK116684)Sequence.

[0110]FIG. 28 shows a D. melanogaster mannosidase II (X77652) Sequence.

[0111]FIG. 29 shows a human mannosidase II (U31520) Sequence.

[0112]FIG. 30 shows a mouse mannosidase II (X61172) Sequence.

[0113]FIG. 31 shows a rat mannosidase II (XM_(—)218816) Sequence.

[0114]FIG. 32 shows a human mannosidase IIx (D55649) Sequence.

[0115]FIG. 33 shows an insect cell mannosidase III (AF005034) Sequence.

[0116]FIG. 34 shows a human lysosomal mannosidase II (NM_(—)000528)Sequence.

[0117]FIG. 35 shows a human cytoplasmic mannosidase II (NM_(—)006715)Sequence.

[0118]FIG. 36 illustrates oligosaccharide intermediates produced usingmannosidase II, mannosidase IIx and mannosidase III activities.

DETAILED DESCRIPTION OF THE INVENTION

[0119] Unless otherwise defined herein, scientific and technical termsused in connection with the present invention shall have the meaningsthat are commonly understood by those of ordinary skill in the art.Further, unless otherwise required by context, singular terms shallinclude pluralities and plural terms shall include the singular. Themethods and techniques of the present invention are generally performedaccording to conventional methods well known in the art. Generally,nomenclatures used in connection with, and techniques of biochemistry,enzymology, molecular and cellular biology, microbiology, genetics andprotein and nucleic acid chemistry and hybridization described hereinare those well-known and commonly used in the art.

[0120] The methods and techniques of the present invention are generallyperformed according to conventional methods well-known in the art and asdescribed in various general and more specific references that are citedand discussed throughout the present specification unless otherwiseindicated. See, e.g., Sambrook et al. Molecular Cloning: A LaboratoryManual, 2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y. (1989); Ausubel et al., Current Protocols in Molecular Biology,Greene Publishing Associates (1992, and Supplements to 2002); Harlow andLane Antibodies: A Laboratory Manual Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (1990); Introduction to Glycobiology,Maureen E. Taylor, Kurt Drickamer, Oxford Univ. Press (2003);Worthington Enzyme Manual, Worthington Biochemical Corp. Freehold, N.J.;Handbook of Biochemistry: Section A Proteins Vol I 1976 CRC Press;Handbook of Biochemistry: Section A Proteins Vol II1976 CRC Press;Essentials of Glycobiology, Cold Spring Harbor Laboratory Press (1999).The nomenclatures used in connection with, and the laboratory proceduresand techniques of, molecular and cellular biology, protein biochemistry,enzymology and medicinal and pharmaceutical chemistry described hereinare those well known and commonly used in the art.

[0121] All publications, patents and other references mentioned hereinare incorporated by reference.

[0122] The following tenns, unless otherwise indicated, shall beunderstood to have the following meanings:

[0123] As used herein, the tenm “N-glycan” refers to an N-linkedoligosaccharide, e.g., one that is attached by anasparagine-N-acetylglucosamine. linkage to an asparagine residue of apolypeptide. N-glycans have a common pentasaecharide core of Man₃GlcNAc₂(“Man” refers to mannose; “Glc” refers to glucose; and “NAc” refers toN-acetyl; GlcNAc refers to N-acetylglucosamine). The term “trimannosecore” used with respect to the N-glycan also refers to the structureMan₃GlcNAc₂ (“Man₃”). N-glycans differ with respect to the number ofbranches (antennae) comprising peripheral sugars (e.g., fucose andsialic acid) that are added to the Man₃ core structure. N-glycans areclassified according to their branched constituents (e.g., high mannose,complex or hybrid).

[0124] A “high mannose” type N-glycan has five or more mannose residues.A “complex” type N-glycan typically has at least one GlcNAc attached tothe 1,3 mannose arm and at least one GlcNAc attached to the 1,6 mannosearm of the trimannose core. Complex N-glycans may also have galactose(“Gal”) residues that are optionally modified with sialic acid orderivatives (“NeuAc”, where “Neu” refers to neuraminic acid and “Ac”refers to acetyl). A complex N-glycan typically has at least one branchthat terminates in an oligosaccharide such as, for example: NeuNAc-;NeuAca2-6GalNAca1-; NeuAca2-3Galb1-3GalNAca1-;NeuAca2-3/6Galb1-4GlcNAcb1-; GlcNAca1-4Galb1-(mucins only);Fuca1-2Galb1-(blood group H). Sulfate esters can occur on galactose,GalNAc, and GlcNAc residues, and phosphate esters can occur on mannoseresidues. NeuAc (Neu: neuraminic acid; Ac:acetyl) can be O-acetylated orreplaced by NeuGl (N-glycolylneuraminic acid). Complex N-glycans mayalso have intrachain substitutions comprising “bisecting” GlcNAc andcore fucose (“Fuc”). A “hybrid” N-glycan has at least one GlcNAc on theterminal of the 1,3 mannose arm of the trimannose core and zero or moremannoses on the 1,6 mannose arm of the trimannose core.

[0125] The term “predominant” or “predominantly” used with respect tothe production of N-glycans refers to a structure which represents themajor peak detected by matrix assisted laser desorption ionization timeof flight mass spectrometry (MALDI-TOF) analysis.

[0126] Abbreviations used herein are of common usage in the art, see,e.g., abbreviations of sugars, above. Other common abbreviations include“PNGase”, which refers to peptide N-glycosidase F (EC 3.2.2.18); “GlcNAcTr” or “GnT,” which refers to N-acetylglucosaminyl Transferase enzymes;“NANA” refers to N-acetylneuraminic acid.

[0127] As used herein, a “humanized glycoprotein” or a “human-likeglycoprotein” refers alternatively to a protein having attached theretoN-glycans having three or less mannose residues, and syntheticglycoprotein intermediates (which are also useful and can be manipulatedfurther in vitro or in vivo). Preferably, glycoproteins producedaccording to the invention contain at least 20 mole %, preferably 20-30mole %, more preferably 30-40 mole %, even more preferably 40-50 mole %and even more preferably 50 100 mole % of the GlcNAcMan₃GlcNAc₂intermediate, at least transiently. This may be achieved, e.g., byengineering a host cell of the invention to express a “better”, i.e., amore efficient glycosylation enzyme. For example, a mannosidase II isselected such that it will have optimal activity under the conditionspresent at the site in the host cell where proteins are glycosylated andis introduced into the host cell preferably by targeting the enzyme to ahost cell organelle where activity is desired.

[0128] The term “enzyme”, when used herein in connection with alteringhost cell glycosylation, refers to a molecule having at least oneenzymatic activity, and includes full-length enzymes, catalyticallyactive fragments, chimerics, complexes, and the like. A “catalyticallyactive fragment” of an enzyme refers to a polypeptide having adetectable level of functional (enzymatic) activity. Enzyme activity is“substantially intracellular” when subsequent processing enzymes havethe ability to produce about 51% of the desired glycoforms in vivo.

[0129] A lower eukaryotic host cell, when used herein in connection withglycosylation profiles, refers to any eukaryotic cell which ordinarilyproduces high mannose containing N-glycans, and thus is meant to includesome animal or plant cells and most typical lower eukaryotic cells,including uni- and multicellular fungal and algal cells.

[0130] As used herein, the term “secretion pathway” refers to theassembly line of various glycosylation enzymes to which a lipid-linkedoligosaccharide precursor and an N-glycan substrate are sequentiallyexposed, following the molecular flow of a nascent polypeptide chainfrom the cytoplasm to the endoplasmic reticulum (ER) and thecompartments of the Golgi apparatus. Enzymes are said to be localizedalong this pathway. An enzyme X that acts on a lipid-linked glycan or anN-glycan before enzyme Y is said to be or to act “upstream” to enzyme Y;similarly, enzyme Y is or acts “downstream” from enzyme X.

[0131] The term “targeting peptide” as used herein refers to nucleotideor amino acid sequences encoding a cellular targeting signal peptidewhich mediates the localization (or retention) of an associated sequenceto sub-cellular locations, e.g., organelles.

[0132] The term “polynucleotide” or “nucleic acid molecule” refers to apolymeric form of nucleotides of at least 10 bases in length. The termincludes DNA molecules (e.g., cDNA or genomic or synthetic DNA) and RNAmolecules (e.g., mRNA or synthetic RNA),.as well as analogs of DNA orRNA containing non-natural nucleotide analogs, non-nativeinternucleoside bonds, or both. The nucleic acid can be in anytopological conformation. For instance, the nucleic acid can besingle-stranded, double-stranded, triple-stranded, quadruplexed,partially double-stranded, branched, hairpinned, circular, or in apadlocked confonnation. The term includes single and double strandedforms of DNA. A nucleic acid molecule of this invention may include bothsense and antisense strands of RNA, cDNA, genomic DNA, and syntheticforms and mixed polymers of the above. They may be modified chemicallyor biochemically or may contain non-natural or derivatized nucleotidebases, as will be readily appreciated by those of skill in the art. Suchmodifications include, for example, labels, methylation, substitution ofone or more of the naturally occurring nucleotides with an analog,intemucleotide modifications such as uncharged linkages (e.g., methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.),charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.),pendent moieties (e.g., polypeptides), intercalators (e.g., acridine,psoralen, etc.), chelators, alkylators, and modified linkages (e.g.,alpha anomeric nucleic acids, etc.) Also included are syntheticmolecules that mimic polynucleotides in their ability to bind to adesignated sequence via hydrogen bonding and other chemicalinteractions. Such molecules are known in the art and include, forexample, those in which peptide linkages substitute for phosphatelinkages in the backbone of the molecule.

[0133] Unless otherwise indicated, a “nucleic acid comprising SEQ IDNO:X” refers to a nucleic acid, at least a portion of which has either(i) the sequence of SEQ ID NO:X, or (ii) a sequence complementary to SEQID NO:X. The choice between the two is dictated by the context. Forinstance, if the nucleic acid is used as a probe, the choice between thetwo is dictated by the requirement that the probe be complementary tothe desired target.

[0134] An “isolated” or “substantially pure” nucleic acid orpolynucleotide (e.g., an RNA, DNA or a mixed polymer) is one which issubstantially separated from other cellular components that naturallyaccompany the native polynucleotide in its natural host cell, e.g.,ribosomes, polymerases, and genomic sequences with which it is naturallyassociated. The term embraces a nucleic acid or polynucleotide that (1)has been removed from its naturally occurring environment, (2) is notassociated with all or a portion of a polynucleotide in which the“isolated polynucleotide” is found in nature, (3) is operatively linkedto a polynucleotide which it is not linked to in nature, or (4) does notoccur in nature. The term “isolated” or “substantially pure” also can beused in reference to recombinant or cloned DNA isolates, chemicallysynthesized polynucleotide analogs, or polynucleotide analogs that arebiologically synthesized by heterologous systems.

[0135] However, “isolated” does not necessarily require that the nucleicacid or polynucleotide so described has itself been physically removedfrom its native environment. For instance, an endogenous nucleic acidsequence in the genome of an organism is deemed “isolated” herein if aheterologous sequence (i.e., a sequence that is not naturally adjacentto this endogenous nucleic acid sequence) is placed adjacent to theendogenous nucleic acid sequence, such that the expression of thisendogenous nucleic acid sequence is altered. By way of example, anon-native promoter sequence can be substituted (e.g., by homologousrecombination) for the native promoter of a gene in the genome of ahuman cell, such that this gene has an altered expression pattern. Thisgene would now become “isolated” because it is separated from at leastsome of the sequences that naturally flank it.

[0136] A nucleic acid is also considered “isolated” if it contains anymodifications that do not naturally occur to the corresponding nucleicacid in a genome. For instance, an endogenous coding sequence isconsidered “isolated” if it contains an insertion, deletion or a pointmutation introduced artificially, e.g., by human intervention. An“isolated nucleic acid” also includes a nucleic acid integrated into ahost cell chromosome at a heterologous site, a nucleic acid constructpresent as an episome. Moreover, an “isolated nucleic acid” can besubstantially free of other cellular material, or substantially free ofculture medium when produced by recombinant techniques, or substantiallyfree of chemical precursors or other chemicals when chemicallysynthesized.

[0137] As used herein, the phrase “degenerate variant” of a referencenucleic acid sequence encompasses nucleic acid sequences that can betranslated, according to the standard genetic code, to provide an aminoacid sequence identical to that translated from the reference nucleicacid sequence.

[0138] The term “percent sequence identity” or “identical” in thecontext of nucleic acid sequences refers to the residues in the twosequences which are the same when aligned for maximum correspondence.The length of sequence identity comparison may be over a stretch of atleast about nine nucleotides, usually at least about 20 nucleotides,more usually at least about 24 nucleotides, typically at least about 28nucleotides, more typically at least about 32 nucleotides, andpreferably at least about 36 or more nucleotides. There are a number ofdifferent algorithms known in the art that can be used to measurenucleotide sequence identity. For instance, polynucleotide sequences canbe compared using FASTA, Gap or Bestfit, which are programs in WisconsinPackage Version 10.0, Genetics Computer Group (GCG), Madison, Wis. FASTAprovides alignments and percent sequence identity of the regions of thebest overlap between the query and search sequences (Pearson, 1990,herein incorporated by reference). For instance, percent sequenceidentity between nucleic acid sequences can be determined using FASTAwith its default parameters (a word size of 6 and the NOPAM factor forthe scoring matrix) or using Gap with its default parameters as providedin GCG Version 6.1, herein incorporated by reference.

[0139] The term “substantial homology” or “substantial similarity,” whenreferring to a nucleic acid or fragment thereof, indicates that, whenoptimally aligned with appropriate nucleotide insertions or deletionswith another nucleic acid (or its complementary strand), there isnucleotide sequence identity in at least about 50%, more preferably 60%of the nucleotide bases, usually at least about 70%, more usually atleast about 80%, preferably at least about 90%, and more preferably atleast about 95%, 96%, 97%, 98% or 99% of the nucleotide bases, asmeasured by any well-known algorithm of sequence identity, such asFASTA, BLAST or Gap, as discussed above.

[0140] Alternatively, substantial homology or similarity exists when anucleic acid or fragment thereof hybridizes to another nucleic acid, toa strand of another nucleic acid, or to the complementary strandthereof, under stringent hybridization conditions. “Stringenthybridization conditions” and “stringent wash conditions” in the contextof nucleic acid hybridization experiments depend upon a number ofdifferent physical parameters. Nucleic acid hybridization will beaffected by such conditions as salt concentration, temperature,solvents, the base composition ofthe hybridizing species, length of thecomplementary regions, and the number of nucleotide base mismatchesbetween the hybridizing nucleic acids, as will be readily appreciated bythose skilled in the art. One having ordinary skill in the art knows howto vary these parameters to achieve a particular stringency ofhybridization.

[0141] In general, “stringent hybridization” is performed at about 25°C. below the thermal melting point (T_(m)) for the specific DNA hybridunder a particular set of conditions. “Stringent washing” is performedat temperatures about 5° C. lower than the T_(m) for the specific DNAhybrid under a particular set of conditions. The T_(m) is thetemperature at which 50% of the target sequence hybridizes to aperfectly matched probe. See Sambrook et al., supra, page 9.51, herebyincorporated by reference. For purposes herein, “high stringencyconditions” are defined for solution phase hybridization as aqueoushybridization (i.e., free of formamide) in 6×SSC (where 20×SSC contains3.0 M NaCl and 0.3 M-sodium citrate), 1% SDS at 65° C. for 8-12 hours,followed by two washes in 0.2×SSC, 0.1% SDS at 65° C. for 20 minutes. Itwill be appreciated by the skilled artisan that hybridization at 65° C.will occur at different rates depending on a number of factors includingthe length and percent identity of the sequences which are hybridizing.

[0142] The term “mutated” when applied to nucleic acid sequences meansthat nucleotides in a nucleic acid sequence may be inserted, deleted orchanged compared to a reference nucleic acid sequence. A singlealteration may be made at a locus (a point mutation) or multiplenucleotides may be inserted, deleted or changed at a single locus. Inaddition, one or more alterations may be made at any number of lociwithin a nucleic acid sequence. A nucleic acid sequence may be mutatedby any method known in the art including but not limited to mutagenesistechniques such as “error-prone PCR” (a process for performing PCR underconditions where the copying fidelity of the DNA polymerase is low, suchthat a high rate of point mutations is obtained along the entire lengthof the PCR product. See, e.g., Leung, D. W., et al., Technique, 1, pp.11-15 (1989) and Caldwell, R. C. & Joyce G. F., PCR Methods Applic., 2,pp. 28-33 (1992)); and “oligonucleotide-directed mutagenesis” (a processwhich enables the generation of site-specific mutations in any clonedDNA segment of interest. See, e.g., Reidhaar-Olson, J. F. & Sauer, R.T., et al., Science, 241, pp. 53-57 (1988)).

[0143] The term “vector” as used herein is intended to refer to anucleic acid molecule capable of transporting another nucleic acid towhich it has been linked. One type of vector is a “plasmid”, whichrefers to a circular double stranded DNA loop into which additional DNAsegments may be ligated. Other vectors include cosmids, bacterialartificial chromosomes (BAC) and yeast artificial chromosomes (YAC).Another type of vector is a viral vector, wherein additional DNAsegments may be ligated into the viral genome (discussed in more detailbelow). Certain vectors are capable of autonomous replication in a hostcell into which they are introduced (e.g., vectors having an origin ofreplication which functions in the host cell). Other vectors can beintegrated into the genome of a host cell upon introduction into thehost cell, and are thereby replicated along with the host genome.Moreover, certain preferred vectors are capable of directing theexpression of genes to which they are operatively linked. Such vectorsare referred to herein as “recombinant expression vectors” (or simply,“expression vectors”).

[0144] “Operatively linked” expression control sequences refers to alinkage in which the expression control sequence is contiguous with thegene of interest to control the gene of interest, as well as expressioncontrol sequences that act in trans or at a distance to control the geneof interest.

[0145] The term “expression control sequence” as used herein refers topolynucleotide sequences which are necessary to affect the expression ofcoding sequences to which they are operatively linked. Expressioncontrol sequences are sequences which control the transcription,post-transcriptional events and translation of nucleic acid sequences.Expression control sequences include appropriate transcriptioninitiation, termination, promoter and enhancer sequences; efficient RNAprocessing signals such as splicing and polyadenylation signals;sequences that stabilize cytoplasmic mRNA; sequences that enhancetranslation efficiency (e.g., ribosome binding sites); sequences thatenhance protein stability; and when desired, sequences that enhanceprotein secretion. The nature of such control sequences differsdepending upon the host organism; in prokaryotes, such control sequencesgenerally include promoter, ribosomal binding site, and transcriptiontermination sequence. The term “control sequences” is intended toinclude, at a minimum, all components whose presence is essential forexpression, and can also include additional components whose presence isadvantageous, for example, leader sequences and fusion partnersequences.

[0146] The term “recombinant host cell” (or simply “host cell”), as usedherein, is intended to refer to a cell into which a nucleic acid such asa recombinant vector has been introduced. It should be understood thatsuch terms are intended to refer not only to the particular subject cellbut to the progeny of such a cell. Because certain modifications mayoccur in succeeding generations due to either mutation or environmentalinfluences, such progeny may not, in fact, be identical to the parentcell, but are still included within the scope of the term “host cell” asused herein. A recombinant host cell may be an isolated cell or cellline grown in culture or may be a cell which resides in a living tissueor organism.

[0147] The term “peptide” as used herein refers to a short polypeptide,e.g., one that is typically less than about 50 amino acids long and moretypically less than about 30 amino acids long. The term as used hereinencompasses analogs and mimetics that mimic structural and thusbiological function.

[0148] The term “polypeptide” as used herein encompasses bothnaturally-occurring and non-naturally-occurring proteins, and fragments,mutants, derivatives and analogs thereof. A polypeptide may be monomericor polymeric. Further, a polypeptide may comprise a number of differentdomains each of which has one or more distinct activities.

[0149] The term “isolated protein” or “isolated polypeptide” is aprotein or polypeptide that by virtue of its origin or source ofderivation (1) is not associated with naturally associated componentsthat accompany it in its native state, (2) when it exists in a puritynot found in nature, where purity can be adjudged with respect to thepresence of other cellular material (e.g., is free of other proteinsfrom the same species) (3) is expressed by a cell from a differentspecies, or (4) does not occur in nature (e.g., it is a fragment of apolypeptide found in nature or it includes amino acid analogs orderivatives not found in nature or linkages other than standard peptidebonds). Thus, a polypeptide that is chemically synthesized orsynthesized in a cellular system different from the cell from which itnaturally originates will be “isolated” from its naturally associatedcomponents. A polypeptide or protein may also be rendered substantiallyfree of naturally associated components by isolation, using proteinpurification techniques well-known in the art. As thus defined,“isolated” does not necessarily require that the protein, polypeptide,peptide or oligopeptide so described has been physically removed fromits native environment.

[0150] The term “polypeptide fragment” as used herein refers to apolypeptide that has an amino-terminal and/or carboxy-terminal deletioncompared to a full-length polypeptide. In a preferred embodiment, thepolypeptide fragment is a contiguous sequence in which the amino acidsequence of the fragment is identical to the corresponding positions inthe naturally-occurring sequence. Fragments typically are at least 5, 6,7, 8, 9 or 10 amino acids long, preferably at least 12, 14, 16 or 18amino acids long, more preferably at least 20 amino acids long, morepreferably at least 25, 30, 35, 40 or 45, amino acids, even morepreferably at least 50 or 60 amino acids long, and even more preferablyat least 70 amino acids long.

[0151] A “modified derivative” refers to polypeptides or fragmentsthereof that are substantially homologous in primary structural sequencebut which include, e.g., in vivo or in vitro chemical and biochemicalmodifications or which incorporate amino acids that are not found in thenative polypeptide. Such modifications include, for example,acetylation, carboxylation, phosphorylation, glycosylation,ubiquitination, labeling, e.g., with radionuclides, and variousenzymatic modifications, as will be readily appreciated by those wellskilled in the art. A variety of methods for labeling polypeptides andof substituents or labels useful for such purposes are well-known in theart, and include radioactive isotopes such as ¹²I, ³²P, ³⁵S, and ³H,ligands which bind to labeled antiligands (e.g., antibodies),fluorophores, chemiluminescent agents, enzymes, and antiligands whichcan serve as specific binding pair members for a labeled ligand. Thechoice of label depends on the sensitivity required, ease of conjugationwith the primer, stability requirements, and available instrumentation.Methods for labeling polypeptides are well-known in the art. See Ausubelet al., Current Potocols in Molecular Biology, Greene PublishingAssociates (1992, and supplement sto 2002) hereby incorporated byreference.

[0152] A “polypeptide mutant” or “mutein” refers to a polypeptide whosesequence contains an insertion, duplication, deletion, rearrangement orsubstitution of one or more amino acids compared to the amino acidsequence of a native or wild type protein. A mutein may have one or moreamino acid point substitutions, in which a single amino acid at aposition has been changed to another amino acid, one or more insertionsand/or deletions, in which one or more amino acids are inserted ordeleted, respectively, in the sequence of the naturally-occurringprotein, and/or truncations of the amino acid sequence at either or boththe amino or carboxy termini. A mutein may have the same but preferablyhas a different biological activity compared to the naturally-occumrngprotein.

[0153] A mutein has at least 70% overall sequence homology to itswild-type counterpart. Even more preferred are muteins having 80%, 85%or 90% overall sequence homology to the wild-type protein. In an evenmore preferred -embodiment, a mutein exhibits 95% sequence identity,even more preferably 97%, even more preferably 98% and even morepreferably 99% overall sequence identity. Sequence homology may bemeasured by any common sequence analysis algorithm, such as Gap orBestfit.

[0154] Preferred amino acid substitutions are those which: (1) reducesusceptibility to proteolysis, (2) reduce susceptibility to oxidation,(3) alter binding affinity for forming protein complexes, (4) alterbinding affinity or enzymatic activity, and (5) confer or modify otherphysicochemical or functional properties of such analogs.

[0155] As used herein, the twenty conventional amino acids and theirabbreviations follow conventional usage. See Immunology—A Synthesis(2^(nd) Edition, E. S. Golub and D. R. Gren, Eds., Sinauer Associates,Sunderland, Mass. (1991)), which is incorporated herein by reference.Stereoisomers (e.g., D-amino acids) of the twenty conventional aminoacids, unnatural amino acids such as α-, α-disubstituted amino acids,N-alkyl amino acids, and other unconventional amino acids may also besuitable components for polypeptides of the present invention. Examplesof unconventional amino acids include: 4-hydroxyproline,γ-carboxyglutamate, ε-N,N,N-trimethyllysine, ε-N-acetyllysine,O-phosphoserine, N-acetylserine, N-forrnylmethionine, 3-methylhistidine,5-hydroxylysine, s-N-methylarginine, and other similar amino acids andimino acids (e.g., 4-hydroxyproline). In the polypeptide notation usedherein, the left-hand direction is the amino terminal direction and theright hand direction is the carboxy-terminal direction, in accordancewith standard usage and convention.

[0156] A protein has “homology” or is “homologous” to a second proteinif the nucleic acid sequence that encodes the protein has a similarsequence to the nucleic acid sequence that encodes the second protein.Alternatively, a protein has homology to a second protein if the twoproteins have “similar” amino acid sequences. (Thus, the term“homologous proteins” is defined to mean that the two proteins havesimilar amino acid sequences). In a preferred embodiment, a homologousprotein is one that exhibits 60% sequence homology to the wild typeprotein, more preferred is 70% sequence homology. Even more preferredare homologous proteins that exhibit 80%, 85% or 90% sequence homologyto the wild type protein. In a yet more preferred embodiment, ahomologous protein exhibits 95%, 97%, 98% or 99% sequence identity. Asused herein, homology between two regions of amino acid sequence(especially with respect to predicted structural similarities) isinterpreted as implying similarity in function.

[0157] When “homologous” is used in reference to proteins or peptides,it is recognized that residue positions that are not identical oftendiffer by conservative amino acid substitutions. A “conservative aminoacid substitution” is one in which an amino acid residue is substitutedby another amino acid residue having a side chain (R group) with similarchemical properties (e.g., charge or hydrophobicity).

[0158] In general, a conservative amino acid substitution will notsubstantially change the functional properties of a protein. In caseswhere two or more amino acid sequences differ from each other byconservative substitutions, the percent sequence identity or degree ofhomology may be adjusted upwards to correct for the conservative natureof the substitution. Means for making this adjustment are well known tothose of skill in the art (see, e.g., Pearson el al., 1994, hereinincorporated by reference).

[0159] The following six groups each contain amino acids that areconservative substitutions for one another: I) Serine (S), Threonine(T); 2) Aspartic Acid (D), Glutamic Acid (E); 3) Asparagine (N),Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (1), Leucine(L), Methionine (M), Alanine (A), Valine (V), and 6) Phenylalanine (F),Tyrosine (Y), Tryptophan (W).

[0160] Sequence homology for polypeptides, which is also referred to aspercent sequence identity, is typically measured using sequence analysissoftware. See, e.g., the Sequence Analysis Software Package of theGenetics Computer Group (GCG), University of Wisconsin BiotechnologyCenter, 910 University Avenue, Madison, Wis. 53705. Protein analysissoftware matches similar sequences using measure of homology assigned tovarious substitutions, deletions and other modifications, includingconservative amino acid substitutions. For instance, GCG containsprograms such as “Gap” and “Bestfit” which can be used with defaultparameters to determine sequence homology or sequence identity betweenclosely related polypeptides, such as homologous polypeptides fromdifferent species of organisms or between a wild type protein and amutein thereof. See, e.g., GCG Version 6.1.

[0161] A preferred algorithm when comparing a inhibitory moleculesequence to a database containing a large number of sequences fromdifferent organisms is the computer program BLAST (Altschul, S. F. etal. (1990) J. Mol. Biol. 215:403-410; Gish and States (1993) NatureGenet. 3:266-272; Madden, T. L. et al. (1996) Meth. Enzymol.266:131-141; Altschul, S. F. et al. (1997) Nucleic Acids Res.25:3389-3402; Zhang, J. and Madden, T. L. (1997) Genome Res. 7:649-656),especially blastp or tblastn (Altschul et al., 1997). Preferredparameters for BLASTp are: Expectation value: 10 (default); Filter: seg(default); Cost to open a gap: 11 (default); Cost to extend a gap: 1(default); Max. alignments: 100 (default); Word size: 11 (default); No.of descriptions: 100 (default); Penalty Matrix: BLOWSUM62.

[0162] The length of polypeptide sequences compared for homology willgenerally be at least about 16 amino acid residues, usually at leastabout 20 residues, more usually at least about 24 residues, typically atleast about 28 residues, and preferably more than about 35 residues.When searching a database containing sequences from a large number ofdifferent organisms, it is preferable to compare amino acid sequences.Database searching using amino acid sequences can be measured byalgorithms other than blastp known in the art. For instance, polypeptidesequences can be compared using FASTA, a program in GCG Version 6.1.FASTA provides alignments and percent sequence identity of the regionsof the best overlap between the query and search sequences (Pearson,1990, herein incorporated by reference). For example, percent sequenceidentity between amino acid sequences can be determined using FASTA withits default parameters (a word size of 2 and the PAM250 scoring matrix),as provided in GCG Version 6. 1, herein incorporated by reference.

[0163] The term “motif” in reference to the conserved regions denotesthe amino acid residues usually found in proteins and conventionallyknown as alanine (Ala or A), valine (Val or V), Jeucine (Leu or L),isoleucine (lie or 1), proline (Pro or P), phenylalanine (Phe or F),tryptophan (Trp or W), methionine (Met or M), glycine (Gly or G), serine(Ser or S), threonine (Thr or T), cysteine (Cys or C), tyrosine (Tyr orY), asparagine (Asn or N), glutamine (Gln or Q), aspartic acid (Asp orD), glutamic acid (Glu or E), lysine (Lys or K), arginine (Arg or R),and histidine (His orH).

[0164] The term “fusion protein” refers to a polypeptide comprising apolypeptide or fragment coupled to heterologous amino acid sequences.Fusion proteins are useful because they can be constructed to containtwo or more desired functional elements from two or more differentproteins. A fusion protein comprises at least 10 contiguous amino acidsfrom a polypeptide of interest, more preferably at least 20 or 30 aminoacids, even more preferably at least 40, 50 or 60 amino acids, yet morepreferably at least 75, 1 00 or 125 amino acids. Fusion proteins can beproduced recombinantly by constructing a nucleic acid sequence whichencodes the polypeptide or a fragment thereof in-frame with a nucleicacid sequence encoding a different protein or peptide and thenexpressing the fusion protein. Alternatively, a fusion protein can beproduced chemically by crosslinking the polypeptide or a fragmentthereof to another protein.

[0165] The term “region” as used herein refers to a physicallycontiguous portion of the primary structure of a biomolecule. In thecase of proteins, a region is defined by a contiguous portion of theamino acid sequence of that protein.

[0166] The term “domain” as used herein refers to a structure of abiomolecule that contributes to a known or suspected function of thebiomolecule. Domains may be co-extensive with regions or portionsthereof; domains may also include distinct, non-contiguous regions of abiomolecule. Examples of protein domains include, but are not limitedto, an Ig domain, an extracellular domain, a transmembrane domain, and acytoplasmic domain.

[0167] As used herein, the term “molecule” means any compound,including, but not limited to, a small molecule, peptide, protein,sugar, nucleotide, nucleic acid, lipid, etc., and such a compound can benatural or synthetic.

[0168] Unless otherwise defined, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention pertains. Exemplary methods andmaterials are described below, although methods and materials similar orequivalent to those described herein can also be used in the practice ofthe present invention and will be apparent to those of skill in the art.All publications and other references mentioned herein are incorporatedby reference in their entirety. In case of conflict, the presentspecification, including definitions, will control. The materials,methods, and examples are illustrative only and not intended to belimiting.

[0169] Throughout this specification and claims, the word “comprise” orvariations such as “comprises” or “comprising”, will be understood toimply the inclusion of a stated integer or group of integers but not theexclusion of any other integer or group of integers.

[0170] Methods for Producing Human-Like Glycoproteins in LowerEukaryotic Host Cells

[0171] The invention provides methods for producing a glycoproteinhaving human-like glycosylation in a non-human eukaryotic host cell. Asdescribed in more detail below, a eukaryotic host cell that does notnaturally express, or which is engineered not to express, one or moreenzymes involved in production of high mannose structures is selected asa starting host cell. Such a selected host cell is engineered to expressone or more enzymes or other factors required to produce human-likeglycoproteins. A desired host strain can be engineered one enzyme ormore than one enzyme at a time. In addition, a nucleic acid moleculeencoding one or more enzymes or activities may be used to engineer ahost strain of the invention. Preferably, a library of nucleic acidmolecules encoding potentially useful enzymes (e.g., chimeric enzymescomprising a catalytically active enzyme fragment ligated in-frame to aheterologous subceleular targeting sequence) is created (e.g., byligation of sub-libraries comprising enzymatic fragments and subcellulartargeting sequences), and a strain having one or more enzymes withoptimal activities or producing the most “human-like” glycoproteins maybe selected by transforming target host cells with one or more membersof the library.

[0172] In particular, the methods described herein enable one to obtain,in vivo, Man₅GlcNAc₂ structures in high yield, at least transiently, forthe purpose of further modifying it to yield complex N-glycans. Asuccessful scheme to obtain suitable Man₅GlcNAc₂ structures inappropriate yields in a host cell, such as a lower eukaryotic organism,generally involves two parallel approaches: (1) reducing high mannosestructures made by endogenous mannosyltransferase activities, if any,and (2) removing 1,2-α-mannose by mannosidases to yield high levels ofsuitable Man₅GlcNAc₂ structures which may be further reacted inside thehost cell to form complex, human-like glycoforms.

[0173] Accordingly, a first step involves the selection or creation of aeukaryotic host cell, e.g., a lower eukaryote, capable of producing aspecific precursor structure of Man₅GlcNAc₂ that is able to accept invivo GlcNAc by the action of a GlcNAc transferase I (“GnTI”). In oneembodiment, the method involves making or using a non-human eukaryotichost cell depleted in a 1,6 mannosyltransferase activity with respect tothe N-glycan on a glycoprotein. Preferably, the host cell is depleted inan initiating 1,6 mannosyltransferase activity (see below). Such a hostcell will lack one or more enzymes involved in the production of highmannose structures which are undesirable for producing human-likeglycoproteins.

[0174] One or more enzyme activities are then introduced into such ahost cell to produce N-glycans within the host cell characterized byhaving at least 30 mol % of the Man₅GlcNAc₂ (“Mans”) carbohydratestructures. Man₅GlcNAc₂ structures are necessary for complex N-glycanformation: Man₅GlcNAc₂ must be formed in vivo in a high yield (e.g., inexcess of 30%), at least transiently, as subsequent mammalian- andhuman-like glycosylation reactions require Man₅GlcNAc₂ or a derivativethereof.

[0175] This step also requires the formation of a particular isomericstructure of Man₅GlcNAc₂ within the cell at a high yield. WhileMan₅GlcNAc₂ structures are necessary for complex N-glycan formation,their presence is by no means sufficient. That is because Man₅GlcNAc₂may occur in different isomeric forms, which may or may not serve as asubstrate for GlcNAc transferase I. As most glycosylation reactions arenot complete, a particular glycosylated protein generally contains arange of different carbohydrate structures (i.e. glycoformns) on itssurface. Thus, the mere presence of trace amounts (i.e., less than 5%)of a particular structure like Man₅GlcNAc₂ is of little practicalrelevance for producing mammalian- or human-like glycoproteins. It isthe formation of a GlcNAc transferase I-accepting Man₅GlcNAc₂intermediate (FIG. 1B) in high yield (i.e., above 30%), which isrequired. The formation of this intermediate is necessary to enablesubsequent in vivo synthesis of complex N-glycans on glycosylatedproteins of interest (target proteins).

[0176] Accordingly, some or all of the Man₅GlcNAc₂ produced by theselected host cell must be a productive substrate for enzyme activitiesalong a mammalian glycosylation pathway, e.g., can serve as a substratefor a GlcNAc transferase I activity in vivo, thereby forming thehuman-like N-glycan intermediate GlcNAcMan₅GlcNAc₂ in the host cell. Ina preferred embodiment, at least 10%, more preferably at least 30% andmost preferably 50% or more of the Man₅GlcNAc₂ intermediate produced inthe host cell of the invention is a productive substrate for GnTI invivo. It is understood that if, for example, GlcNAcMan₅GlcNAc₂ isproduced at 10% and Man₅GlcNAc₂ is produced at 25% on a target protein,that the total amount of transiently produced Man₅GlcNAc₂ is 35% becauseGlcNAcMan₅GlcNAc₂ is a product of Man₅GlcNAc₂.

[0177] One of ordinary skill in the art can select host cells fromnature, e.g., existing fungi or other lower eukaryotes that producesignificant levels of Man₅GlcNAc₂ in vivo. As yet, however, no lowereukaryote has been shown to provide such structures in vivo in excess of1.8% of the total N-glycans (see e.g. Maras et al., 1997, Eur. J.Biochem. 249, 701-707). Alternatively, such host cells may begenetically engineered to produce the Man₅GlcNAc₂ structure in vivo.Methods such as those described in U.S. Pat. No. 5,595,900 may be usedto identify the absence or presence of particular glycosyltransferases,mannosidases and sugar nucleotide transporters in a target host cell ororganism of interest.

[0178] Inactivation of Undesirable Host Cell Glycosylation Enzymes

[0179] The methods of the invention are directed to making host cellswhich produce glycoproteins having altered, and preferably human-like,N-glyean structures. In a preferred embodiment, the methods are directedto making host cells in which oligosaccharide precursors are enriched inMan₅GlcNAc₂. Preferably, a eukaryotic host cell is used that does notexpress one or more enzymes involved in the production of high mannosestructures. Such a host cell may be found in nature or may beengineered, e.g., starting with or derived from one of many such mutantsalready described in yeasts. Thus, depending on the selected host cell,one or a number of genes that encode enzymes known to be characteristicof non-human glycosylation reactions will have to be deleted. Such genesand their corresponding proteins have been extensively characterized ina number of lower eukaryotes (e.g., S.cerevisiae, T. reesei, A. nidulansetc.), thereby providing a list of known glycosyltransferases in lowereukaryotes, their activities and their respective genetic sequence.These genes are likely to be selected from the group ofmannosyltransferases e.g. 1,3 mannosyltransferases (e.g. MNN1 inS.cerevisiae) (Graham, 1991), 1,2 mannosyltransferases (e.g. KTR/KREfamily from S.cerevisiae), 1,6 mannosyltransferases (OCH1 fromS.cerevisiae), mannosylphosphate transferases and their regulators (MNN4and MNN6 from S.cerevisiae) and additional enzymes that are involved inaberrant, i.e. non human, glycosylation reactions. Many of these geneshave in fact been deleted individually giving rise to viable phenotypeswith altered glycosylation profiles. Examples are shown in Table 1(above).

[0180] Preferred lower eukaryotic host cells of the invention, asdescribed herein to exemplify the required manipulation steps, arehypermannosylation-minus (och1) mutants of Pichia pastoris or K.lactis.Like other lower eukaryotes, P.pasioris processes Man₉GlcNAc₂ structuresin the ER with an α-1,2-mannosidase to yield Man₈GlcNAc₂ (FIG. 1A).Through the action of several mannosyltransferases, this structure isthen converted to hypermannosylated structures (Man_(>9)GlcNAc₂), alsoknown as mannans. In addition, it has been found that P.pastoris is ableto add non-terminal phosphate groups, through the action ofmannosylphosphate transferases, to the carbohydrate structure. Thisdiffers from the reactions performed in mammalian cells, which involvethe removal rather than addition of mannose sugars. It is of particularimportance to eliminate the ability of the eukaryotic host cell, e.g.,fungus, to hypernannosylate an existing Man₈GlcNAc₂ structure. This canbe achieved by either selecting for a host cell that does nothypernannosylate or by genetically engineering such a cell.

[0181] Genes that are involved in the hypermnannosylation process havebeen identified, e.g., in Pichia pastoris, and by creating mutations inthese genes, one can reduce the production of “undesirable” glycoforms.Such genes can be identified by homology to existingmannosyltransferases or their regulators (e.g., OCH1, MNN4, MNN6, MANN)found in other lower eukaryotes such as C. albicans, Pichia angusta orS.cerevisiae or by mutagenizing the host strain and selecting for aglycosylation phenotype with reduced mannosylation. Based on homologiesamongst known mannosyltransferases and mannosylphosphate transferases,one may either design PCR primers (examples of which are shown in Table2, SEQ ID Nos: 60-91 are additional examples of primers), or use genesor gene fragments encoding such enzymes as probes to identify homologsin DNA libraries of the target or a related organism. Alternatively, onemay identify a functional homolog having mannosyltransferase activity byits ability to complement particular glycosylation phenotypes in relatedorganisms. TABLE 2 PCR Primers Target Gene(s) PCR primer A PCR primer Bin P. pastoris Homologs ATGGCGAAGGCAG TTAGTCCTTCCA 1,6-mannosyl- OCH1 S.cerevisiae, ATGGCAGT ACTTCCTTC transferase Pichia albicans TAYTGGMGNGTNGGCRTCNCCCCAN 1,2 mannosyl- KTR/KRE family, ARCYNGAYATHAA CKYTCRTAtransferases S. cerevisiae

[0182] To obtain the gene or genes encoding 1,6-mannosyltransferaseactivity in P. pastoris, for example, one would carry out the followingsteps: OCH1 mutants of S.cerevisiae are temperature sensitive and areslow growers at elevated temperatures. One can thus identify functionalhomologs of OCH1 in P.pasioris by complementing an OCH1 mutant ofS.cerevisiae with a P.pastoris DNA or cDNA library. Mutants ofS.cerevisiae are available, e.g., from Stanford University and arecommercially available from ResGen, an Invitrogen Corp. (Carlsbad,Calif.). Mutants that display a normal growth phenotype at elevatedtemperature, after having been transformed with a P.pastoris DNAlibrary, are likely to carry an OCH1 homolog of P. pastoris. Such alibrary can be created by partially digesting chromosomal DNA ofP.pastoris with a suitable restriction enzyme and, after inactivatingthe restriction enzyme, ligating the digested DNA into a suitablevector, which has been digested with a compatible restriction enzyme.

[0183] Suitable vectors include, e.g., pRS314, a low copy (CEN6/ARS4)plasmid based on pBluescript containing the Trp1 marker (Sikorski, R.S., and Hieter, P.,1989, Genetics 122, pg 19-27) and pFL44S, a high copy(2μ) plasmid based on a modified pUC19 containing the URA3 marker(Bonneaud, N., et al., 1991, Yeast 7, pg. 609-615). Such vectors arecommonly used by academic researchers and similar vectors are availablefrom a number of different vendors (e.g., Invitrogen (Carlsbad, Calif.);Pharmacia (Piscataway, N.J.); New England Biolabs (Beverly, Mass.)).Further examples include pYES/GS, 2μ origin of replication based yeastexpression plasmid from Invitrogen, or Yep24 cloning vehicle from NewEngland Biolabs.

[0184] After ligation of the chromosomal DNA and the vector, one maytransform the DNA library into a strain of S.cerevisiae with a specificmutation and select for the correction of the corresponding phenotype.After sub-cloning and sequencing the DNA fragment that is able torestore the wild-type phenotype, one may use this fragment to eliminatethe activity of the gene product encoded by OCH1 in P.pastoris using invivo mutagenesis and/or recombination techniques well-known to thoseskilled in the art.

[0185] Alternatively, if the entire genomic sequence of a particularhost cell, e.g., fungus, of interest is known, one may identify suchgenes simply by searching publicly available DNA databases, which areavailable from several sources, such as NCBI, Swissprot. For example, bysearching a given genomic sequence or database with sequences from aknown 1,6 mannosyltransferase gene (e.g., OCH1 from S.cerevisiae), onecan identify genes of high homology in such a host cell genome which may(but do not necessarily) encode proteins that have1,6-mannosyltransferase activity. Nucleic acid sequence homology aloneis not enough to prove, however, that one has identified and isolated ahomolog encoding an enzyme having the same activity. To date, forexample, no data exist to show that an OCH1 deletion in P.pastoriseliminates the crucial initiating 1,6-mannosyltransferase activity.(Martinet et al. Biotech. Letters 20(12) (December 1998): 1171-1177;Contreras et al. WO 02/00856 A2). Thus, no data prove that theP.pastoris OCH1 gene homolog actually encodes that function. Thatdemonstration is provided for the first time herein.

[0186] Homologs to several S.cerevisiae mannosyltransferases have beenidentified in P.pastoris using these approaches. Homologous genes oftenhave similar functions to genes involved in the mannosylation ofproteins in S.cerevisiae and thus their deletion may be used tomanipulate the glycosylation pattern in P.pastoris or, by analogy, inany other host cell, e.g., fungus, plant, insect or animal cells, withsimilar glycosylation pathways.

[0187] The creation of gene knock-outs, once a given target genesequence has been determined, is a well-established technique in the artand can be carried out by one of ordinary skill in the art (see, e.g.,R. Rothstein, (1991) Methods in Enzymology, vol. 194, p. 281). Thechoice of a host organism may be influenced by the availability of goodtransformation and gene disruption techniques.

[0188] If several mannosyltransferases are to be knocked out, the methoddeveloped by Alani and Kleckner, (Genetics 116:541-545 (1987)), forexample, enables the repeated use of a selectable marker, e.g., the URA3marker in yeast, to sequentially eliminate all undesirable endogenousmannosyltransferase activity. This technique has been refined by othersbut basically involves the use of two repeated DNA sequences, flanking acounter selectable marker. For example: URA3 may be used as a marker toensure the selection of a transformants that have integrated aconstruct. By flanking the URA3 marker with direct repeats one may firstselect for transformants that have integrated the construct and havethus disrupted the target gene. After isolation of the transformants,and their characterization, one may counter select in a second round forthose that are resistant to 5-fluoroorotic acid (5-FOA). Colonies thatare able to survive on plates containing 5-FOA have lost the URA3 markeragain through a crossover event involving the repeats mentioned earlier.This approach thus allows for the repeated use of the same marker andfacilitates the disruption of multiple genes without requiringadditional markers. Similar techniques for sequential elimination ofgenes adapted for use in another eukaryotic host cells with otherselectable and counter-selectable markers may also be used.

[0189] Eliminating specific mannosyltransferases, such as 1,6mannosyltransferase (OCH1) or mannosylphosphate transferases (MNN6, orgenes complementing lbd mutants) or regulators (MNN4) in P.pastorisenables one to create engineered strains of this organism whichsynthesize primarily Man₈GlcNAc₂ and which can be used to further modifythe glycosylation pattern to resemble more complex glycoform structures,e.g., those produced in mammalian, e.g., human cells. A preferredembodiment of this method utilizes DNA sequences encoding biochemicalglycosylation activities to eliminate similar or identical biochemicalfunctions in P. pastoris to modify the glycosylation structure ofglycoproteins produced in the genetically altered P.pastoris strain.

[0190] Methods used to engineer the glycosylation pathway in yeasts asexemplified herein can be used in filamentous fungi to produce apreferred substrate for subsequent modification. Strategies formodifying glycosylation pathways in A.niger and other filamentous fungi,for example, can be developed using protocols analogous to thosedescribed herein for engineering strains to produce human-likeglycoproteins in yeast. Undesired gene activities involved in 1,2mannosyltransferase activity, e.g., KTR/KRE homologs, are modified oreliminated. A filamentous fungus, such as Aspergillus, is a preferredhost because it lacks the 1,6 mannosyltransferase activity and as such,one would not expect a hypermannosylating gene activity, e.g. OCH1, inthis host. By contrast, other desired activities (e.g.,α-1,2-mannosidase, UDP-GlcNAc transporter, glycosyltransferase (GnT),galactosyltransferase (GalT) and sialyltransferase (ST)) involved inglycosylation are introduced into the host using the targeting methodsof the invention.

[0191] Engineering or Selecting Hosts Having Diminished Initiating α-1,6Mannosyltransferase Activity

[0192] In a preferred embodiment, the method of the invention involvesmaking or using a host cell which is diminished or depleted in theactivity of an initiating α-1,6-mannosyltransferase, i.e., an initiationspecific enzyme that initiates outer chain mannosylation on the α-1,3arm of the Man₃GlcNAc₂ core structure. In S.cerevisiae, this enzyme isencoded by the OCH1 gene. Disruption of the OCH1 gene in S.cerevisiaeresults in a phenotype in which N-linked sugars completely lack thepoly-mannose outer chain. Previous approaches for obtainingmammalian-type glycosylation in fungal strains have requiredinactivation of OCH1 (see, e.g., Chiba et al. (1998) J. Biol. Chem.273:26298-304). Disruption of the initiating α-1,6-mannosyltransferaseactivity in a host cell of the invention may be optional, however(depending on the selected host cell), as the Och1p enzyme requires anintact Man₈GlcNAc₂ for efficient mannose outer chain initiation. Thus,host cells selected or produced according to this invention whichaccumulate oligosaccharides having seven or fewer mannose residues mayproduce hypoglycosylated N-glycans that will likely be poor substratesfor Och1p (see, e.g., Nakayama et al. (1997) FEBS Lett. 412(3):547-50).

[0193] The OCH1 gene was cloned from P.pastoris (Example 1) and K.lactis(Example 9), as described. The nucleic acid and amino acid sequences ofthe OCH1 gene from K.lactis are set forth in SEQ ID NOS: 3 and 4. Usinggene-specific primers, a construct was made from each clone to deletethe OCH1 gene from the genome of Ppastoris and K.lactis (Examples 1 and9, respectively). Host cells depleted in initiatingα-1,6-mannosyltransferase activity and engineered to produce N-glycanshaving a Man₅GlcNAc₂ carbohydrate structure were thereby obtained (see,e.g., Examples 4 and 9).

[0194] Thus, in another embodiment, the invention provides an isolatednucleic acid molecule having a nucleic acid sequence comprising orconsisting of at least forty-five, preferably at least 50, morepreferably at least 60 and most preferably 75 or more nucleotideresidues of the K.lactis OCH1 gene (SEQ ID NO: 3), and homologs,variants and derivatives thereof. The invention also provides nucleicacid molecules that hybridize under stringent conditions to theabove-described nucleic acid molecules. Similarly, isolated polypeptides(including muteins, allelic variants, fragments, derivatives, andanalogs) encoded by the nucleic acid molecules of the invention areprovided. Also provided are vectors, including expression vectors, whichcomprise the above nucleic acid molecules of the invention, as describedfurther herein. Similarly, host cells transformed with the nucleic acidmolecules or vectors of the invention are provided.

[0195] The invention further provides methods of making or using anon-human eukaryotic host cell diminished or depleted in an alg geneactivity (i.e., alg activities, including equivalent enzymaticactivities in non-fungal host cells) and introducing into the host cellat least one glycosidase activity. In a preferred embodiment, theglycosidase activity is introduced by causing expression of one or moremannosidase activities within the host cell, for example, by activationof a mannosidase activity, or by expression from a nucleic acid moleculeof a mannosidase activity, in the host cell.

[0196] In yet another embodiment, the invention provides a method forproducing a human-like glycoprotein in a non-human host, wherein theglycoprotein comprises an N-glycan having at least two GlcNAcs attachedto a trimannose core structure.

[0197] Expression of Class 2 Mannosidases in Lower Eukaryotes

[0198] The present invention additionally provides a method for makingmore human-like glycoproteins in lower eukaryotic host cells byexpressing a gene encoding a catalytically active Class 2 mannosidases(EC. 3.2.1.114) (homologs, variants, derivatives and catalyticallyactive fragment thereof).

[0199] Using known techniques in the art, gene-specific primers aredesigned to complement the homologous regions of the Class 2 mannosidasegenes (e.g. D.melanogaster α-mannosidase II) in order to PCR amplify themannosidase gene.

[0200] Through the expression of an active Class 2 mannosidase in a cellfrom a nucleic acid encoding the Class 2 mannosidase a host cell (e.g.P. pastoris) is engineered to produce more human-like glycoproteins(see, e.g., Examples 17-25).

[0201] In one aspect of the invention, a method is provided forproducing a human-like glycoprotein in a lower eukaryote (e.g. P.pastoris) by constructing a library of α-mannosidase II enzymes. In apreferred embodiment, a sub-library of D.melanogaster α-mannosidase IIsequences (e.g. Genbank Accession No. X77652) is fused to a sub-libraryof S.cerevisiae MNN2 targeting peptide sequences. In a more preferredembodiment of the invention, a fusion construct comprising D.melanogaster Mannosidase II Δ74/MNN2(s) is transformed into a P.pastoris host producing GlcNAcMan₅GlcNAc₂. See Choi et al. Proc NatlAcad Sci U S A. 2003 Apr. 29;100(9):5022-7 and WO 02/00879, whichdisclose methods for making human-like glycoproteins in lower eukaryoteshaving the above N-glycan structure, which is now designated P. pastorisYSH-1.

[0202] In another embodiment, a Golgi α-mannosidase II sequence isselected from, rat, mouse, human, worms, plants and insects. Suchsequences are available in databases such as Swissprot and Genbank. Forexample, sequences for the following genes were found in Genbank:Arabidopsis thaliana Mannosidase II (NM_(—)121499); C. elegansMannosidase II (NM_(—)073594); Ciona intestinalis mannosidase II(AK116684); Drosophila melanogaster mannosidase II (X77652); humanmannosidase II (U31520); mouse mannosidase II (X61172); rat mannosidaseII (XM_(—)218816); human mannosidase IIx (D55649); insect cellmannosidase III (AF005034); human lysosomal mannosidase II(NM_(—)000528); and human cytosolic mannosidase II (NM_(—)006715) (FIGS.25-35, SEQ ID NOs: 49-59, respectively). Because of the high sequencesimilarity and the presence of the Manα1,3 and Manα1,6 activity,cytoplasmic mannosidase II and lysosomal mannosidase II will becollectively referred to herein as Class 2 mannosidases.

[0203] Other mannosidases that exhibit the Golgi α-mannosidase IIactivity include, inter alia, insect mannosidase III (AF005034) andhuman mannosidase IIx (D55649). As such, these mannosidases may also beused to generate a combinatorial DNA library of catalytically activeenzymes.

[0204] In another aspect of the invention, a sub-library of targetingpeptide sequences (leaders) selected from the group consisting ofSaccharomyces GLS1, Saccharomyces MNS1, Saccharomyces SEC 12, PichiaSEC, Pichia OCH1, Saccharomyces MNN9, Saccharomyces VAN1, SaccharomycesANP1, Saccharomyces HOC1, Saccharomyces MN 10, Saccharomyces MNN11,Saccharomyces MNT1, Pichia D2, Pichia D9, Pichia J3, Saccharomyces KTR1,Saccharomyces KTR2, Kluyveromyces GnTI, Saccharomyces MNN2,Saccharomyces MNN5, Saccharomyces YUR1, Saccharomyces MNN1, andSaccharomyces MNN6 are fused to sequences encoding catalytically activemannosidase II domains. The combination of the leader/catalytic domainlibrary is illustrated in Table 11 (Example 14).

[0205] The Golgi α-mannosidase II fusion constructs generated accordingto the present invention display the α1,3 and α1,6 mannosidase trimmingactivity. For example, the catalytically active mannosidase II fusionconstruct cleaves the Manα1,3 and Manα1,6 glycosidic linkages present onGlcNAcMan₅GlcNAc₂ to GlcNAcMan₃GlcNAc₂ in P. pastoris YSH-1. In anotherexample, a catalytically active mannosidase IIx fusion construct cleavesthe Manα1,3 and Manα1,6 glycosidic linkages present on Man₆GlcNAc₂ toMan₄GlcNAc₂.

[0206] Class 2 Mannosidase Hydrolysis of Glycosidic Linkage

[0207] The present invention also encompasses the mechanism in which thecatalytically active domain of Class 2 enzymes hydrolyze the Manα1,3and/or Manα1,6 glycosidic linkages on an oligosaccharide e.g.GlcNAcMan₅GlcNAc₂ structure to produce GlcNAcMan₃GlcNAc₂, a desiredintermediate for further N-glycan processing in a lower eukaryote. In afirst embodiment, the hydrolysis of the glycosidic linkages occurssequentially. The enzyme hydrolyzes at least one glycosidic linkage andconformationally rotates to hydrolyze the other glycosidic linkage. In asecond embodiment, the hydrolysis of the glycosidic linkages occurssimultaneously. The intermediate produced is a substrate for furtherGolgi processing wherein other glycosylation enzymes such asN-acetylglucosaminyltransferases (GnTs), galactosyltransferases (GalTs)and sialyltransferases (STs) can subsequently modify it to produce adesired glycoform. FIG. 36A illustrates the oligosaccharideintermediates (e.g. GlcNAcMan₃GlcNAc₂, GlcNAcMan₄GlcNAc₂) produced viathe mannosidase II pathway and FIG. 36B illustrates the oligosaccharideintermediates (e.g. Man₄GlcNAc₂, Man₅GlcNAc₂) produced via themannosidase IIx pathway.

[0208] Conserved Regions of the Mannosidase II Enzymes

[0209] It is a feature of the present invention to express sequencesencoding conserved regions of the mannosidase II enzyme. The presentinvention provides isolated nucleic acid molecules that comprise theconserved regions of the mannosidase II gene from various sourcesincluding insect, mammals, plants and worms.

[0210] Several full-length nucleic acid sequences encoding themannosidase II enzyme have been identified and sequenced. Themannosidase II enzyme sequences are set forth in SEQ ID NO: 49 throughSEQ ID NO: 59. An alignment of known mannosidase II sequences (i.e.,Drosophila melanogaster aligned to other insect, animal and plantsequences) shows a highly conserved motif between amino acids 144-166and amino acids 222-285 (FIG. 23). Accordingly, in another aspect, theinvention relates to a method for providing to a host cell a nucleicacid encoding a Class 2 mannosidase enzyme activity wherein the nucleicacid is characterized by having the above conserved mannosidase IIregions.

[0211] Moreover, the sequence alignment further reveals several highlyconserved cystine-cystine disulfide bridges between amino acids 338-345and amino acids 346-360 as shown in FIG. 23. These disulfide bridges mayplay an integral part in substrate binding and recognition, e.g., bymaintaining protein architecture.

[0212] The present invention also provides catalytically activefragments of Class 2 mannosidases comprising conserved amino acidsequence regions, especially a first amino acid sequence consisting of23 amino acid residues having the following sequence: 144 Leu Lys ValPhe Val Val Pro His Ser (SEQ ID NO: 5) His Asn Asp Pro Gly Trp Ile GlnThr Phe Glu Glu Tyr Try.

[0213] In another preferred embodiment, the amino acid residue atposition 145 of the first sequence is selected from the group consistingof K E Q N and Y.

[0214] In another preferred embodiment, the amino acid residue atposition 146 of the first sequence is selected from the group consistingof V and I.

[0215] In another preferred embodiment, the amino acid residue atposition 147 of the first sequence is selected from the group consistingof F I H and L.

[0216] In another preferred embodiment, the amino acid residue atposition 148 of the first sequence is selected from the group consistingof V I L and T.

[0217] In another preferred embodiment, the amino acid residue atposition 149 of the first sequence is selected from the group consistingof V I L and D.

[0218] In another preferred embodiment, the amino acid residue atposition 150 of the first sequence is selected from the group consistingof P and R.

[0219] In another preferred embodiment, the amino acid residue atposition 151 of the first sequence is selected from the group consistingof H and L.

[0220] In another preferred embodiment, the amino acid residue atposition 152 of the first sequence is selected from the group consistingof S T and G.

[0221] In another preferred embodiment, the amino acid residue atposition 153 of the first sequence is selected from the group consistingof H and E.

[0222] In another preferred embodiment, the amino acid residue atposition 154 of the first sequence is selected from the group consistingof N C D and R.

[0223] In another preferred embodiment, the amino acid residue atposition 156 of the first sequence is selected from the group consistingof P and V.

[0224] In another preferred embodiment, the amino acid residue atposition 157 of the first sequence is selected from the group consistingof G and R

[0225] In another preferred embodiment, the amino acid residue atposition 158 of the first sequence is selected from the group consistingof W and L

[0226] In another preferred embodiment, the amino acid residue atposition 159 of the first sequence is selected from the group consistingof I L K and T.

[0227] In another preferred embodiment, the amino acid residue atposition 160 of the first sequence is selected from the group consistingof Q M K and L.

[0228] In another preferred embodiment, the amino acid residue atposition 161 of the first sequence is selected from the group consistingof T and Y.

[0229] In another preferred embodiment, the amino acid residue atposition 162 of the first sequence is selected from the group consistingof F and V.

[0230] In another preferred embodiment, the amino acid residue atposition 163 of the first sequence is selected from the group consistingof E D and N.

[0231] In another preferred embodiment, the amino acid residue atposition 164 of the first sequence is selected from the group consistingof E K D R Q and V.

[0232] In another preferred embodiment, the amino acid residue atposition 165 of the first sequence is selected from the group consistingof Y and A.

[0233] In another preferred embodiment, the amino acid residue atposition 166 of the first sequence is selected from the group consistingof Y F and C.

[0234] The present invention further provides a catalytically activefragment of a Class 2 mannosidase comprising conserved amino acidsequence regions, especially a second amino acid sequence consisting of57 amino acid residues having the following sequence: 222 Glu Phe ValThr Gly Gly Trp Val Met (SEQ ID NO: 6) Pro Asp Glu Ala Asn Ser Trp ArgAsn Val Leu Leu Gln Leu Thr Glu Gly Gln Thr Trp Leu Lys Gln Phe Met AsnVal Thr Pro Thr Ala Ser Trp Ala Ile Asp Pro Phe Gly His Ser Pro Thr MetPro Tyr Ile Leu.

[0235] In another preferred embodiment, the amino acid residue atposition 222 of the first sequence is selected from the group consistingof E and R.

[0236] In another preferred embodiment, the amino acid residue atposition 223 of the first sequence is selected from the group consistingof F I and S.

[0237] In another preferred embodiment, the amino acid residue atposition 224 of the first sequence is selected from the group consistingof V A T and F

[0238] In another preferred embodiment, the amino acid residue atposition 225 of the first sequence is selected from the group consistingof T G N and Q.

[0239] In another preferred embodiment, the amino acid residue atposition 226 of the first sequence is selected from the group consistingof G and A.

[0240] In another preferred embodiment, the amino acid residue atposition 227 of the first sequence is selected from the group consistingof G and L.

[0241] In another preferred embodiment, the amino acid residue atposition 228 of the first sequence is selected from the group consistingof W and Y.

[0242] In another preferred embodiment, the amino acid residue atposition 229 of the first sequence is selected from the group consistingof V and T.

[0243] In another preferred embodiment, the amino acid residue atposition 230 of the first sequence is selected from the group consistingof M and A.

[0244] In another preferred embodiment, the amino acid residue atposition 231 of the first sequence is selected from the group consistingof P T and N.

[0245] In another preferred embodiment, the amino acid residue atposition 232 of the first sequence is selected from the group consistingof D and Q.

[0246] In another preferred embodiment, the amino acid residue atposition 233 of the first sequence is selected from the group consistingof E and M.

[0247] In another preferred embodiment, the amino acid residue atposition 234 of the first sequence is selected from the group consistingof A and V.

[0248] In another preferred embodiment, the amino acid residue atposition 235 of the first sequence is selected from the group consistingof N T C and A.

[0249] In another preferred embodiment, the amino acid residue atposition 236 of the first sequence is selected from the group consistingof S A P T and V.

[0250] In another preferred embodiment, the amino acid residue atposition 237 of the first sequence is selected from the group consistingof H and C.

[0251] In another preferred embodiment, the amino acid residue atposition 238 of the first sequence is selected from the group consistingof W Y I and D.

[0252] In another preferred embodiment, the amino acid residue atposition 239 of the first sequence is selected from the group consistingof R H F Y G and P.

[0253] In another preferred embodiment, the amino acid residue atposition 240 of the first sequence is selected from the group consistingof N S and A.

[0254] In another preferred embodiment, the amino acid residue atposition 241 of the first sequence is selected from the group consistingof V M L I and Q.

[0255] In another preferred embodiment, the amino acid residue atposition 242 of the first sequence is selected from the group consistingof L I V and P.

[0256] In another preferred embodiment, the amino acid residue atposition 243 of the first sequence is selected from the group consistingof L T G D and E.

[0257] In another preferred embodiment, the amino acid residue atposition 244 of the first sequence is selected from the group consistingof Q E and T.

[0258] In another preferred embodiment, the amino acid residue atposition 245 of the first sequence is selected from the group consistingof L M and F.

[0259] In another preferred embodiment, the amino acid residue atposition 246 of the first sequence is selected from the group consistingof T F I A and P.

[0260] In another preferred embodiment, the amino acid residue atposition 247 of the first sequence is selected from the group consistingof E L and V.

[0261] In another preferred embodiment, the amino acid residue atposition 248 of the first sequence is selected from the group consistingof G and A.

[0262] In another preferred embodiment, the amino acid residue atposition 249 of the first sequence is selected from the group consistingof Q H P M N and L.

[0263] In another preferred embodiment, the amino acid residue atposition 250 of the first sequence is selected from the group consistingof T E P Q M H R and A.

[0264] In another preferred embodiment, the amino acid residue atposition 251 of the first sequence is selected from the group consistingof W P F and L.

[0265] In another preferred embodiment, the amino acid residue atposition 252 of the first sequence is selected from the group consistingof L I V and A.

[0266] In another preferred embodiment, the amino acid residue atposition 253 of the first sequence is selected from the group consistingof K Q R E N and S.

[0267] In another preferred embodiment, the amino acid residue atposition 254 of the first sequence is selected from the group consistingof Q N R K D and T.

[0268] In another preferred embodiment, the amino acid residue atposition 255 of the first sequence is selected from the group consistingof F H N and T.

[0269] In another preferred embodiment, the amino acid residue atposition 256 of the first sequence is selected from the group consistingof M I L and F.

[0270] In another preferred embodiment, the amino acid residue atposition 257 of the first sequence is selected from the group consistingof N and G.

[0271] In another preferred embodiment, the amino acid residue atposition 258 of the first sequence is selected from the group consistingof V A G and H.

[0272] In another preferred embodiment, the amino acid residue atposition 259 of the first sequence is selected from the group consistingof T I K V R and G.

[0273] In another preferred embodiment, the amino acid residue atposition 260 of the first sequence is selected from the group consistingof P and G.

[0274] In another preferred embodiment, the amino acid residue atposition 261 of the first sequence is selected from the group consistingof T Q R K and E.

[0275] In another preferred embodiment, the amino acid residue atposition 262 of the first sequence is selected from the group consistingof A S N T and V.

[0276] In another preferred embodiment, the amino acid residue atposition 263 of the first sequence is selected from the group consistingof S H G A and Q.

[0277] in another preferred embodiment, the amino acid residue atposition 264 of the first sequence is selected from the group consistingof W and H.

[0278] In another preferred embodiment, the amino acid residue atposition 265 of the first sequence is selected from the group consistingof A S H and T.

[0279] In another preferred embodiment, the amino acid residue atposition 266 of the first sequence is selected from the group consistingof I and V.

[0280] In another preferred embodiment, the amino acid residue atposition 267 of the first sequence is selected from the group consistingof D and H.

[0281] In another preferred embodiment, the amino acid residue atposition 268 of the first. sequence is selected from the groupconsisting of P and A.

[0282] In another preferred embodiment, the amino acid residue atposition 269 of the first sequence is selected from the group consistingof F and T.

[0283] In another preferred embodiment, the amino acid residue atposition 271 of the first sequence is selected from the group consistingof H L and Y.

[0284] In another preferred embodiment, the amino acid residue atposition 272 of the first sequence is selected from the group consistingof S T G and C.

[0285] , In another preferred embodiment, the amino acid residue atposition 273 of the first sequence is selected from the group consistingof P S A R and H.

[0286] In another preferred embodiment, the amino acid residue atposition 274 of the first sequence is selected from the group consistingof T S E and 1.

[0287] In another preferred embodiment, the amino acid residue atposition 275 of the first sequence is selected from the group consistingof M V Q and D.

[0288] In another preferred embodiment, the amino acid residue atposition 276 of the first sequence is selected from the group consistingof P A and T.

[0289] In another preferred embodiment, the amino acid residue atposition 277 of the first sequence is selected from the group consistingof Y H S and A.

[0290] In another preferred embodiment, the amino acid residue atposition 278 of the first sequence is selected from the group consistingof I L and W.

[0291] In another preferred embodiment, the amino acid residue atposition 279 of the first sequence is selected from the group consistingof L and F.

[0292] In another preferred embodiment, the amino acid residue atposition 280 of the first sequence is selected from the group consistingof Q T R K N D A and W.

[0293] In another preferred embodiment, the amino acid residue atposition 281 of the first sequence is selected from the group consistingof K S R Q and P.

[0294] In another preferred embodiment, the amino acid residue atposition 282 of the first sequence is selected from the group consistingof S A and M.

[0295] In another preferred embodiment, the amino acid residue atposition 283 of the first sequence is selected from the group consistingof G N and K.

[0296] In another preferred embodiment, the amino acid residue atposition 284 of the first sequence is selected from the group consistingof F I and L.

[0297] In another preferred embodiment, the amino acid residue atposition 285 of the first sequence is selected from the group consistingof K T S E and D.

[0298] The present invention also provides a catalytically activefragment of a Class 2 mannosidase comprising conserved amino acidsequence regions, especially a third amino acid sequence consisting of33 amino acid residues having the following sequence: 325 His Met MetPro Phe Tyr Ser Tyr Asp (SEQ ID NO: 7) Ile Pro His Thr Cys Gly Pro AspPro Arg Ile Cys Cys Gln Phe Asp Phe Arg Arg Met Pro Gly Gly Arg.

[0299] In another preferred embodiment, the amino acid residue atposition 325 of the first sequence is selected from the group consistingof H P and S.

[0300] In another preferred embodiment, the amino acid residue atposition 326 of the first sequence is selected from the group consistingof M I L N T and R.

[0301] In another preferred embodiment, the amino acid residue atposition 327 of the first sequence is selected from the group consistingof M Q A and Y.

[0302] In another preferred embodiment, the amino acid residue atposition 328 of the first sequence is selected from the group consistingof P and D.

[0303] In another preferred embodiment, the amino acid residue atposition 329 of the first sequence is selected from the group consistingof F L and G.

[0304] In another preferred embodiment, the amino acid residue atposition 330 of the first sequence is selected from the group consistingof Y D F and L.

[0305] In another preferred embodiment, the amino acid residue atposition 331 of the first sequence is selected from the group consistingof S I T and Y.

[0306] In another preferred embodiment, the amino acid residue atposition 332 of the first sequence is selected from the group consistingof Y G and S.

[0307] In another preferred embodiment, the amino acid residue atposition 333 of the first sequence is selected from the group consistingof D S V and R.

[0308] In another preferred embodiment, the amino acid residue atposition 334 of the first sequence is selected from the group consistingof I V and L.

[0309] In another preferred embodiment, the amino acid residue atposition 335 of the first sequence is selected from the group consistingof P K and Q.

[0310] In another preferred embodiment, the amino acid residue atposition 336 of the first sequence is selected from the group consistingof H S N and E.

[0311] In another preferred embodiment, the amino acid residue atposition 337 of the first sequence is selected from the group consistingof T G and F.

[0312] In another preferred embodiment, the amino acid residue atposition 338 of the first sequence is selected from the group consistingof C Y and A.

[0313] In another preferred embodiment, the amino acid residue atposition 339 of the first sequence is selected from the group consistingof G N and C.

[0314] In another preferred embodiment, the amino acid residue atposition 340 of the first sequence is selected from the group consistingof P and R.

[0315] In another preferred embodiment, the amino acid residue atposition 341 of the first sequence is selected from the group consistingof D E H P and G.

[0316] In another preferred embodiment, the amino acid residue atposition 342 of the first sequence is selected from the group consistingof P R and Q.

[0317] In another preferred embodiment, the amino acid residue atposition 343 of the first sequence is selected from the group consistingof K S A N and F.

[0318] In another preferred embodiment, the amino acid residue atposition 344 of the first sequence is selected from the group consistingof V I and L.

[0319] In another preferred embodiment, the amino acid residue atposition 345 of the first sequence is selected from the group consistingof C and P.

[0320] In another preferred embodiment, the amino acid residue atposition 346 of the first sequence is selected from the group consistingof C L W and V.

[0321] In another preferred embodiment, the amino acid residue atposition 347 of the first sequence is selected from the group consistingof Q S D and G.

[0322] In another preferred embodiment, the amino acid residue atposition 348 of the first sequence is selected from the group consistingof F V and G.

[0323] In another preferred embodiment, the amino acid residue atposition 349 of the first sequence is selected from the group consistingof D L and T.

[0324] In another preferred embodiment, the amino acid residue atposition 350 of the first sequence is selected from the group consistingof F C and W.

[0325] In another preferred embodiment, the amino acid residue atposition 351 of the first sequence is selected from the group consistingof R K A and V.

[0326] In another preferred embodiment, the amino acid residue atposition 352 of the first sequence is selected from the group consistingof R K D and E.

[0327] In another preferred embodiment, the amino acid residue atposition 353 of the first sequence is selected from the group consistingof M L I and Q.

[0328] In another preferred embodiment, the amino acid residue atposition 354 of the first sequence is selected from the group consistingof G P R and D.

[0329] In another preferred embodiment, the amino acid residue atposition 355 of the first sequence is selected from the group consistingof S E G

[0330] In another preferred embodiment, the amino acid residue atposition 356 of the first sequence is selected from the group consistingof F G and N.

[0331] In another preferred embodiment, the amino acid residue atposition 357 of the first sequence is selected from the group consistingof G R K and L.

[0332] The present invention further provides a catalytically activefragment of a Class 2 mannosidase comprising conserved amino acidsequence regions, especially a fourth amino acid sequence consisting of28 amino acid residues having the following sequence: 380 Leu Leu LeuAsp Gln Tyr Arg Lys Lys (SEQ ID NO: 8) Ser Glu Leu Phe Arg Thr Asn ValLeu Leu Ile Pro Leu Gly Asp Asp Phe Arg Tyr.

[0333] In another preferred embodiment, the amino acid residue atposition 380 of the first sequence is selected from the group consistingof L M I K T and Y.

[0334] In another preferred embodiment, the amino acid residue atposition 381 of the first sequence is selected from the group consistingof L I F and C.

[0335] In another preferred embodiment, the amino acid residue atposition 382 of the first sequence is selected from the group consistingof V Y L I and S.

[0336] In another preferred embodiment, the amino acid residue atposition 383 of the first sequence is selected from the group consistingof D E N and K.

[0337] In another preferred embodiment, the amino acid residue atposition 384 of the first sequence is selected from the group consistingof Q E V and F.

[0338] In another preferred embodiment, the amino acid residue atposition 385 of the first sequence is selected from the group consistingof W Y and A.

[0339] In another preferred embodiment, the amino acid residue atposition 386 of the first sequence is selected from the group consistingof R D T and L.

[0340] In another preferred embodiment, the amino acid residue atposition 387 of the first sequence is selected from the group consistingof K R A and P.

[0341] In another preferred embodiment, the amino acid residue atposition 388 of the first sequence is selected from the group consistingof K I Q and D.

[0342] In another preferred embodiment, the amino acid residue atposition 389 of the first sequence is selected from the group consistingof A S G and T.

[0343] In another preferred embodiment, the amino acid residue atposition 390 of the first sequence is selected from the group consistingof E Q R K T S and F.

[0344] In another preferred embodiment, the amino acid residue atposition 391 of the first sequence is selected from the group consistingof L Y and G.

[0345] In another preferred embodiment, the amino acid residue atposition 392 of the first sequence is selected from the group consistingof Y F and T.

[0346] In another preferred embodiment, the amino acid residue atposition 393 of the first sequence is selected from the group consistingof R P and S.

[0347] In another preferred embodiment, the amino acid residue atposition 394 of the first sequence is selected from the group consistingof T N S H and A.

[0348] In another preferred embodiment, the amino acid residue atposition 395 of the first sequence is selected from the group consistingof N S K D and Q.

[0349] In another preferred embodiment, the amino acid residue atposition 396 of the first sequence is selected from the group consistingof V T H and L.

[0350] In another preferred embodiment, the amino acid residue atposition 397 of the first sequence is selected from the group consistingof L I V T and P.

[0351] In another preferred embodiment, the amino acid residue atposition 398 of the first sequence is selected from the group consistingof L F V and Q.

[0352] In another preferred embodiment, the amino acid residue atposition 399 of the first sequence is selected from the group consistingof I Q V A and M.

[0353] In another preferred embodiment, the amino acid residue atposition 400 of the first sequence is selected from the group consistingof P I T and M.

[0354] In another preferred embodiment, the amino acid residue atposition 401 of the first sequence is selected from the group consistingof L M and H.

[0355] In another preferred embodiment, the amino acid residue atposition 403 of the first sequence is selected from the group consistingof D S and C.

[0356] In another preferred embodiment, the amino acid residue atposition 404 of the first sequence is selected from the group consistingof D and G.

[0357] In another preferred embodiment, the amino acid residue atposition 405 of the first sequence is selected from the group consistingof F and I.

[0358] In another preferred embodiment, the amino acid residue atposition 406 of the first sequence is selected from the group consistingof R and Q.

[0359] In another preferred embodiment, the amino acid residue atposition 407 of the first sequence is selected from the group consistingof F Y and R.

[0360] The present invention also provides a catalytically activefragment of a Class 2 mannosidase comprising conserved amino acidsequence regions, especially a fifth amino acid sequence consisting of12 amino acid residues having the following sequence: 438 Gln Phe GlyThr Leu Ser Asp Tyr Phe (SEQ ID NO: 9) Asp Ala Leu.

[0361] In another preferred embodiment, the amino acid residue atposition 438 of the first sequence is selected from the group consistingof Q K L and H.

[0362] In another preferred embodiment, the amino acid residue atposition 439 of the first sequence is selected from the group consistingof F and Y.

[0363] In another preferred embodiment, the amino acid residue atposition 440 of the first sequence is selected from the group consistingof G S and P.

[0364] In another preferred embodiment, the amino acid residue atposition 441 of the first sequence is selected from the group consistingof T and P.

[0365] In another preferred embodiment, the amino acid residue atposition 442 of the first sequence is selected from the group consistingof L P and G.

[0366] In another preferred embodiment, the amino acid residue atposition 443 of the first sequence is selected from the group consistingof Q S E L A and D.

[0367] In another preferred embodiment, the amino acid residue atposition 444 of the first sequence is selected from the group consistingof E D C and S.

[0368] In another preferred embodiment, the amino acid residue atposition 445 of the first sequence is selected from the group consistingof Y and F.

[0369] In another preferred embodiment, the amino acid residue atposition 446 of the first sequence is selected from the group consistingof F L and G.

[0370] In another preferred embodiment, the amino acid residue atposition 447 of the first sequence is selected from the group consistingof D K R N W and M.

[0371] In another preferred embodiment, the amino acid residue atposition 448 of the first sequence is selected from the group consistingof A K T E and Q.

[0372] In another preferred embodiment, the amino acid residue atposition 449 of the first sequence is selected from the group consistingof V L M and G.

[0373] The present invention also provides a catalytically activefragment of a Class 2 mannosidase comprising conserved amino acidsequence regions, especially a sixth amino acid sequence consisting of14 amino acid residues having the following sequence: 463 Leu Ser GlyAsp Phe Phe Thr Tyr (SEQ ID NO: 10) Ala Asp Arg Ser Asp His.

[0374] In another preferred embodiment, the amino acid residue atposition 463 of the first sequence is selected from the group consistingof L F and K.

[0375] In another preferred embodiment, the amino acid residue atposition 464 of the first sequence is selected from the group consistingof S K H and D.

[0376] In another preferred embodiment, the amino acid residue atposition 465 of the first sequence is selected from the group consistingof G D and V.

[0377] In another preferred embodiment, the amino acid residue atposition 466 of the first sequence is selected from the group consistingof D and A.

[0378] In another preferred embodiment, the amino acid residue atposition 467 of the first sequence is selected from the group consistingof F and N.

[0379] In another preferred embodiment, the amino acid residue atposition 468 of the first sequence is selected from the group consistingof F and N.

[0380] In another preferred embodiment, the amino acid residue atposition 469 of the first sequence is selected from the group consistingof T S V P and R.

[0381] In another preferred embodiment, the amino acid residue atposition 470 of the first sequence is selected from the group consistingof Y and D.

[0382] In another preferred embodiment, the amino acid residue atposition 471 of the first sequence is selected from the group consistingof A S and K.

[0383] In another preferred embodiment, the amino acid residue atposition 472 of the first sequence is selected from the group consistingof D and G.

[0384] In another preferred embodiment, the amino acid residue atposition 473 of the first sequence is selected from the group consistingof R I and G.

[0385] In another preferred embodiment, the amino acid residue atposition 474 of the first sequence is selected from the group consistingof S D E Q F P and A.

[0386] In another preferred embodiment, the amino acid residue atposition 475 of the first sequence is selected from the group consistingof D Q S H and N.

[0387] In another preferred embodiment, the amino acid residue atposition 476 of the first sequence is selected from the group consistingof N H D E and Q.

[0388] The present invention further provides a catalytically activefragment of a Class 2 mannosidase comprising conserved amino acidsequence regions, especially a seventh amino acid sequence consisting of20 amino acid residues having the following sequence: 477 Tyr Trp SerGly Tyr Tyr Thr Ser (SEQ ID NO: 11) Arg Pro Phe Tyr Arg Arg Met Asp ArgVal Leu Glu.

[0389] In another preferred embodiment, the amino acid residue atposition 477 of the first sequence is selected from the group consistingof Y and F.

[0390] In another preferred embodiment, the amino acid residue atposition 478 of the first sequence is selected from the group consistingof W and G.

[0391] In another preferred embodiment, the amino acid residue atposition 479 of the first sequence is selected from the group consistingof S T and F.

[0392] In another preferred embodiment, the amino acid residue atposition 481 of the first sequence is selected from the group consistingof Y and D.

[0393] In another preferred embodiment, the amino acid residue atposition 482 of the first sequence is selected from the group consistingof Y F and G.

[0394] In another preferred embodiment, the amino acid residue atposition 483 of the first sequence is selected from the group consistingof T V S and G.

[0395] In another preferred embodiment, the amino acid residue atposition 484 of the first sequence is selected from the group consistingof S T and G.

[0396] In another preferred embodiment, the amino acid residue atposition 485 of the first sequence is selected from the group consistingof R and G.

[0397] In another preferred embodiment, the amino acid residue atposition 487 of the first sequence is selected from the group consistingof Y F A and T.

[0398] In another preferred embodiment, the amino acid residue atposition 488 of the first sequence is selected from the group consistingof H Y F L and Q.

[0399] In another preferred embodiment, the amino acid residue atposition 489 of the first sequence is selected from the group consistingof K and T.

[0400] In another preferred embodiment, the amino acid residue atposition 490 of the first sequence is selected from the group consistingof R Q S M A and I.

[0401] In another preferred embodiment, the amino acid residue atposition 491 of the first sequence is selected from the group consistingof M L Q V and Y.

[0402] In another preferred embodiment, the amino acid residue atposition 492 of the first sequence is selected from the group consistingof D E and A.

[0403] In another preferred embodiment, the amino acid residue atposition 494 of the first sequence is selected from the group consistingof V I Q and L.

[0404] In another preferred embodiment, the amino acid residue atposition 495 of the first sequence is selected from the group consistingof L M F S and K.

[0405] In another preferred embodiment, the amino acid residue atposition 496 of the first sequence is selected from the group consistingof M Q E Y and R.

[0406] The present invention also provides a catalytically activefragment of a Class 2 mannosidase comprising conserved amino acidsequence regions, especially a eighth amino acid sequence consisting of27 amino acid residues having the following sequence: 524 Ala Arg ArgGlu Leu Gly Leu Phe (SEQ ID NO: 12) Gln His His Asp Ala Ile Thr Gly ThrAla Arg Asp His Val Val Val Asp Tyr Gly.

[0407] In another preferred embodiment, the amino acid residue atposition 524 of the first sequence is selected from the group consistingof A L and W.

[0408] In another preferred embodiment, the amino acid residue atposition 525 of the first sequence is selected from the group consistingof R N and V.

[0409] In another preferred embodiment, the amino acid residue atposition 526 of the first sequence is selected from the group consistingof R Q E and G.

[0410] In another preferred embodiment, the amino acid residue atposition 527 of the first sequence is selected from the group consistingof E A T and N.

[0411] In another preferred embodiment, the amino acid residue atposition 528 of the first sequence is selected from the group consistingof L and M.

[0412] In another preferred embodiment, the amino acid residue atposition 529 of the first sequence is selected from the group consistingof S G A and F.

[0413] In another preferred embodiment, the amino acid residue atposition 530 of the first sequence is selected from the group consistingof L and V.

[0414] In another preferred embodiment, the amino acid residue atposition 531 of the first sequence is selected from the group consistingof F L and E.

[0415] In another preferred embodiment, the amino acid residue atposition 532 of the first sequence is selected from the group consistingof Q and L.

[0416] In another preferred embodiment, the amino acid residue atposition 534 of the first sequence is selected from the group consistingof H and N.

[0417] In another preferred embodiment, the amino acid residue atposition 535 of the first sequence is selected from the group consistingof D and G.

[0418] In another preferred embodiment, the amino acid residue atposition 536 of the first sequence is selected from the group consistingof G A and T.

[0419] In another preferred embodiment, the amino acid residue atposition 537 of the first sequence is selected from the group consistingof I V and Y.

[0420] In another preferred embodiment, the amino acid residue atposition 538 of the first sequence is selected from the group consistingof T and S.

[0421] In another preferred embodiment, the amino acid residue atposition 539 of the first sequence is selected from the group consistingof G and T.

[0422] In another preferred embodiment, the amino acid residue atposition 540 of the first sequence is selected from the group consistingof T and H.

[0423] In another preferred embodiment, the amino acid residue atposition 541 of the first sequence is selected from the group consistingof A and S.

[0424] In another preferred embodiment, the amino acid residue atposition 542 of the first sequence is selected from the group consistingof K R and Q.

[0425] In another preferred embodiment, the amino acid residue atposition 543 of the first sequence is selected from the group consistingof T D E S Q and I.

[0426] In another preferred embodiment, the amino acid residue atposition 544 of the first sequence is selected from the group consistingof H A W Y S and K.

[0427] In another preferred embodiment, the amino acid residue atposition 545 of the first sequence is selected from the group consistingof V and K.

[0428] In another preferred embodiment, the amino acid residue atposition 546 of the first sequence is selected from the group consistingof V M A and G.

[0429] In another preferred embodiment, the amino acid residue atposition 547 of the first sequence is selected from the group consistingof V L Q and N.

[0430] In another preferred embodiment, the amino acid residue atposition 548 of the first sequence is selected from the group consistingof D and R.

[0431] In another preferred embodiment, the amino acid residue atposition 549 of the first sequence is selected from the group consistingof Y and E.

[0432] In another preferred embodiment, the amino acid residue atposition 550 of the first sequence is selected from the group consistingof E G A and C.

[0433] The present invention also provides a catalytically activefragment of a Class 2 mannosidase comprising conserved amino acidsequence regions, especially a ninth amino acid sequence consisting of11 amino acid residues having the following sequence: 788 Gly Ala TyrLeu Phe Leu Pro Asp (SEQ ID NO: 13) Gly Glu Ala.

[0434] In another preferred embodiment, the amino acid residue atposition 789 of the first sequence is selected from the group consistingof A and W.

[0435] In another preferred embodiment, the amino acid residue atposition 790 of the first sequence is selected from the group consistingof Y and D.

[0436] In another preferred embodiment, the amino acid residue atposition 791 of the first sequence is selected from the group consistingof L I and V.

[0437] In another preferred embodiment, the amino acid residue atposition 792 of the first sequence is selected from the group consistingof F and M.

[0438] In another preferred embodiment, the amino acid residue atposition 793 of the first sequence is selected from the group consistingof L K M R and D.

[0439] In another preferred embodiment, the amino acid residue atposition 794 of the first sequence is selected from the group consistingof P and Y.

[0440] In another preferred embodiment, the amino acid residue atposition 795 of the first sequence is selected from the group consistingof N D A and H.

[0441] In another preferred embodiment, the amino acid residue atposition 796 of the first sequence is selected from the group consistingof G N Y Q and L.

[0442] In another preferred embodiment, the amino acid residue atposition 797 of the first sequence is selected from the group consistingof P E Q N and D.

[0443] In another preferred embodiment, the amino acid residue atposition 798 of the first sequence is selected from the group consistingof A G S K and T.

[0444] The present invention further provides a catalytically activefragment of a Class 2 mannosidase comprising conserved amino acidsequence regions, especially a tenth amino acid sequence consisting of14 amino acid residues having the following sequence: 867 Phe Tyr ThrAsp Leu Asn Gly Phe Gln Met Gln Lys Arg Arg. (SEQ ID NO: 14)

[0445] In another preferred embodiment, the amino acid residue atposition 867 of the first sequence is selected from the group consistingof F T and Y.

[0446] In another preferred embodiment, the amino acid residue atposition 868 of the first sequence is selected from the group consistingof Y F S and E.

[0447] In another preferred embodiment, the amino acid residue atposition 869 of the first sequence is selected from the group consistingof T I and S.

[0448] In another preferred embodiment, the amino acid residue atposition 870 of the first sequence is selected from the group consistingof D and Q.

[0449] In another preferred embodiment, the amino acid residue atposition 871 of the first sequence is selected from the group consistingof L T Q S and F.

[0450] In another preferred embodiment, the amino acid residue atposition 872 of the first sequence is selected from the group consistingof N S and G.

[0451] In another preferred embodiment, the amino acid residue atposition 873 of the first sequence is selected from the group consistingof G T and H.

[0452] In another preferred embodiment, the amino acid residue atposition 874 of the first sequence is selected from the group consistingof L M F AY and R.

[0453] In another preferred embodiment, the amino acid residue atposition 875 of the first sequence is selected from the group consistingof Q R and E.

[0454] In another preferred embodiment, the amino acid residue atposition 876 of the first sequence is selected from the group consistingof F M V I Y and R.

[0455] In another preferred embodiment, the amino acid residue atposition 877 of the first sequence is selected from the group consistingof I Q S L and P.

[0456] In another preferred embodiment, the amino acid residue atposition 878 of the first sequence is selected from the group consistingof K P R E and T.

[0457] In another preferred embodiment, the amino acid residue atposition 879 of the first sequence is selected from the group consistingof R and H.

[0458] In another preferred embodiment, the amino acid residue atposition 880 of the first sequence is selected from the group consistingof R M T E V and Y.

[0459] The present invention further provides a catalytically activefragment of a Class 2 mannosidase comprising conserved amino acidsequence regions, especially a eleventh amino acid sequence consistingof 66 amino acid residues having the following sequence: 904 Lys Leu ProLeu Gln Ala Asn Tyr Tyr Pro Met Pro Ser Met Ala Tyr Ile Gln Asp (SEQ IDNO: 15) Ala Asn Thr Arg Leu Thr Leu Leu Thr Gly Gln Pro Leu Gly Val SerSer Leu Ala Ser Gly Gln Leu Glu Val Met Leu Asp Arg Arg Leu Met Ser AspAsp Asn Arg Gly Leu Gly Gln Gly Val Leu Asp Asn Lys.

[0460] In another preferred embodiment, the amino acid residue atposition 904 of the first sequence is selected from the group consistingof N T Q E and K.

[0461] In another preferred embodiment, the amino acid residue atposition 905 of the first sequence is selected from the group consistingof T R K H S Q M and F.

[0462] In another preferred embodiment, the amino acid residue atposition 906 of the first sequence is selected from the group consistingof R Q and G.

[0463] In another preferred embodiment, the amino acid residue atposition 907 of the first sequence is selected from the group consistingof L M and F.

[0464] In another preferred embodiment, the amino acid residue atposition 908 of the first sequence is selected from the group consistingof T S and A.

[0465] In another preferred embodiment, the amino acid residue atposition 909 of the first sequence is selected from the group consistingof L 1 and V.

[0466] In another preferred embodiment, the amino acid residue atposition 910 of the first sequence is selected from the group consistingof L H and M.

[0467] In another preferred embodiment, the amino acid residue atposition 911 of the first sequence is selected from the group consistingof T S and N.

[0468] In another preferred embodiment, the amino acid residue atposition 912 of the first sequence is selected from the group consistingof G A R N and D.

[0469] In another preferred embodiment, the amino acid residue atposition 913 of the first sequence is selected from the group consistingof Q H R and C.

[0470] In another preferred embodiment, the amino acid residue atposition 914 of the first sequence is selected from the group consistingof P A S and K.

[0471] In another preferred embodiment, the amino acid residue atposition 915 of the first sequence is selected from the group consistingof L Q and Y.

[0472] In another preferred embodiment, the amino acid residue atposition 917 of the first sequence is selected from the group consistingof G V and A.

[0473] In another preferred embodiment, the amino acid residue atposition 918 of the first sequence is selected from the group consistingof S and A.

[0474] In another preferred embodiment, the amino acid residue atposition 919 of the first sequence is selected from the group consistingof S and A.

[0475] In another preferred embodiment, the amino acid residue atposition 920 of the first sequence is selected from the group consistingof L M and Y.

[0476] In another preferred embodiment, the amino acid residue atposition 921 of the first sequence is selected from the group consistingof A S G K E R and V.

[0477] In another preferred embodiment, the amino acid residue atposition 922 of the first sequence is selected from the group consistingof S N D E P and R.

[0478] In another preferred embodiment, the amino acid residue atposition 924 of the first sequence is selected from the group consistingof E Q W R and S.

[0479] In another preferred embodiment, the amino acid residue atposition 925 of the first sequence is selected from the group consistingof L and I.

[0480] In another preferred embodiment, the amino acid residue atposition 926 of the first sequence is selected from the group consistingof E and L.

[0481] In another preferred embodiment, the amino acid residue atposition 927 of the first sequence is selected from the group consistingof I V L and S.

[0482] In another preferred embodiment, the amino acid residue atposition 928 of the first sequence is selected from the group consistingof M I F V and L.

[0483] In another preferred embodiment, the amino acid residue atposition 929 of the first sequence is selected from the group consistingof Q L M V and S.

[0484] In another preferred embodiment, the amino acid residue atposition 930 of the first sequence is selected from the group consistingof D H and L.

[0485] In another preferred embodiment, the amino acid residue atposition 931 of the first sequence is selected from the group consistingof R and L.

[0486] In another preferred embodiment, the amino acid residue atposition 933 of the first sequence is selected from the group consistingof L T and A.

[0487] In another preferred embodiment, the amino acid residue atposition 934 of the first sequence is selected from the group consistingof A S M V L and P.

[0488] In another preferred embodiment, the amino acid residue atposition 935 of the first sequence is selected from the group consistingof S Q R Y and K.

[0489] In another preferred embodiment, the amino acid residue atposition 936 of the first sequence is selected from the group consistingof D and A.

[0490] In another preferred embodiment, the amino acid residue atposition 937 of the first sequence is selected from the group consistingof D and P.

[0491] In another preferred embodiment, the amino acid residue atposition 938 of the first sequence is selected from the group consistingof E N G F and D.

[0492] In another preferred embodiment, the amino acid residue atposition 939 of the first sequence is selected from the group consistingof R and A.

[0493] In another preferred embodiment, the amino acid residue atposition 940 of the first sequence is selected from the group consistingof G and T.

[0494] In another preferred embodiment, the amino acid residue atposition 941 of the first sequence is selected from the group consistingof L V I and A.

[0495] In another preferred embodiment, the amino acid residue atposition 942 of the first sequence is selected from the group consistingof G Q E S and D.

[0496] In another preferred embodiment, the amino acid residue atposition 943 of the first sequence is selected from the group consistingof Q E and T.

[0497] In another preferred embodiment, the amino acid residue atposition 944 of the first sequence is selected from the group consistingof G and P.

[0498] In another preferred embodiment, the amino acid residue atposition 945 of the first sequence is selected from the group consistingof V L I and R.

[0499] In another preferred embodiment, the amino acid residue atposition 946 of the frst sequence is selected from the group consistingof L R K H Q M and V.

[0500] In another preferred embodiment, the amino acid residue atposition 947 of the first sequence is selected from the group consistingof D and E.

[0501] In another preferred embodiment, the amino acid residue atposition 948 of the first sequence is selected from the group consistingof N and F.

[0502] In another preferred embodiment, the amino acid residue atposition 949 of the first sequence is selected from the group consistingof K L R G and T.

[0503] In another preferred embodiment, the amino acid residue atposition 950 of the first sequence is selected from the group consistingof P R I A S and Y.

[0504] In another preferred embodiment, the amino acid residue atposition 951 of the first sequence is selected from the group consistingof V T M G and A.

[0505] In another preferred embodiment, the amino acid residue atposition 952 of the first sequence is selected from the group consistingof L V C A P T.

[0506] In another preferred embodiment, the amino acid residue atposition 953 of the first sequence is selected from the group consistingof H A N E V F W and M.

[0507] In another preferred embodiment, the amino acid residue atposition 954 of the first sequence is selected from the group consistingof I H R L S V Q and P.

[0508] In another preferred embodiment, the amino acid residue atposition 955 of the first sequence is selected from the group consistingof Y F N R and H.

[0509] In another preferred embodiment, the amino acid residue atposition 956 of the first sequence is selected from the group consistingof R V H W G and K.

[0510] In another preferred embodiment, the amino acid residue atposition 957 of the first sequence is selected from the group consistingof L I R and G.

[0511] In another preferred embodiment, the amino acid residue atposition 958 of the first sequence is selected from the group consistingof V L M H and S.

[0512] In another preferred embodiment, the amino acid residue atposition 959 of the first sequence is selected from the group consistingof L I A F.

[0513] In another preferred embodiment, the amino acid residue atposition 960 of the first sequence is selected from the group consistingof E V and Q.

[0514] In another preferred embodiment, the amino acid residue atposition 961 of the first sequence is selected from the group consistingof K P R S L and D.

[0515] In another preferred embodiment, the amino acid residue atposition 962 of the first sequence is selected from the group consistingof V M R W N L and A.

[0516] In another preferred embodiment, the amino acid residue atposition 963 of the first sequence is selected from the group consistingof N S T I P D and G.

[0517] In another preferred embodiment, the amino acid residue atposition 964 of the first sequence is selected from the group consistingof N S L V A G and T.

[0518] In another preferred embodiment, the amino acid residue atposition 965 of the first sequence is selected from the group consistingof C S M G V I Q and A.

[0519] In another preferred embodiment, the amino acid residue atposition 966 of the first sequence is selected from the group consistingof V S N A T and Q.

[0520] In another preferred embodiment, the amino acid residue atposition 967 of the first sequence is selected from the group consistingof R G P M T A and D.

[0521] In another preferred embodiment, the amino acid residue atposition 968 of the first sequence is selected from the group consistingof P N E K and A.

[0522] In another preferred embodiment, the amino acid residue atposition 969 of the first sequence is selected from the group consistingof S K V E A K and Y.

[0523] In another preferred embodiment, the amino acid residue atposition 970 of the first sequence is selected from the group consistingof K Q E S R and A.

[0524] In another preferred embodiment, the amino acid residue atposition 971 of the first sequence is selected from the group consistingof L E Q D K N and G.

[0525] In another preferred embodiment, the amino acid residue atposition 972 of the first sequence is selected from the group consistingof H E S K T and N.

[0526] In another preferred embodiment, the amino acid residue atposition 973 of the first sequence is selected from the group consistingof P R S K and N.

[0527] In another preferred embodiment, the amino acid residue atposition 974 of the first sequence is selected from the group consistingof A V T L P and Y.

[0528] In another preferred embodiment, the amino acid residue atposition 975 of the first sequence is selected from the group consistingof G S A R and Q.

[0529] In another preferred embodiment, the amino acid residue atposition 976 of the first sequence is selected from the group consistingof Y F N and V.

[0530] In another preferred embodiment, the amino acid residue atposition 977 of the first sequence is selected from the group consistingof L H and P.

[0531] In another preferred embodiment, the amino acid residue atposition 978 of the first sequence is selected from the group consistingof T S and L.

[0532] In another preferred embodiment, the amino acid residue atposition 979 of the first sequence is selected from the group consistingof S H L M and Q.

[0533] In another preferred embodiment, the amino acid residue atposition 980 of the first sequence is selected from the group consistingof A V L and T.

[0534] In another preferred embodiment, the amino acid residue atposition 981 of the first sequence is selected from the group consistingof A G S V and L.

[0535] In another preferred embodiment, the amino acid residue atposition 982 of the first sequence is selected from the group consistingof H Y D L and P.

[0536] In another preferred embodiment, the amino acid residue atposition 983 of the first sequence is selected from the group consistingof K L M I Q Y and A.

[0537] In another preferred embodiment, the amino acid residue atposition 984 of the first sequence is selected from the group consistingof A T S I L and P.

[0538] In another preferred embodiment, the amino acid residue atposition 985 of the first sequence is selected from the group consistingof S T G and E.

[0539] In another preferred embodiment, the amino acid residue atposition 986 of the first sequence is selected from the group consistingof Q W M S A R and P.

[0540] In another preferred embodiment, the amino acid residue atposition 987 of the first sequence is selected from the group consistingof S Y F L E H M and A.

[0541] In another preferred embodiment, the amino acid residue atposition 988 of the first sequence is selected from the group consistingof L M F V and P.

[0542] In another preferred embodiment, the amino acid residueatposition 989 of the first sequence is selected from the group consistingof L H N and A.

[0543] In another preferred embodiment, the amino acid residue atposition 990 of the first sequence is selected from the group consistingof D Y T A and H.

[0544] In another preferred embodiment, the amino acid residue atposition 991 of the first sequence is selected from the group consistingof P and S.

[0545] In another preferred embodiment, the amino acid residue atposition 992 of the first sequence is selected from the group consistingof L P A F V I Q and W.

[0546] In another preferred embodiment, the amino acid residue atposition 993 of the first sequence is selected from the group consistingof D VIL I R N and S.

[0547] In another preferred embodiment, the amino acid residue atposition 994 of the first sequence is selected from the group consistingof K V A P and T.

[0548] In another preferred embodiment, the amino acid residue atposition 995 of the first sequence is selected from the group consistingof F M L and Y.

[0549] In another preferred embodiment, the amino acid residue atposition 996 of the first sequence is selected from the group consistingof I P V A S and L.

[0550] In another preferred embodiment, the amino acid residue atposition 997 of the first sequence is selected from the group consistingof F G V L N A and P.

[0551] In another preferred embodiment, the amino acid residue atposition 998 of the first sequence is selected from the group consistingof A D A S N K and G.

[0552] In another preferred embodiment, the amino acid residue atposition 999 of the first sequence is selected from the group consistingof E A K R G T and S.

[0553] Expression of Class III Mannosidases in Lower Eukaryotes

[0554] The present invention also provides that a mannosidase havingsubstrate specificity to Manα1,2/Manα1,3/Manα1,6 be introduced into alower eukaryote host.

[0555] In one embodiment, a class III mannosidase capable of hydrolyzingManα1,2/Manα1,3/Manα1,6 glycosidic linkages is expressed in a lowereukaryotic host. By expressing Class III mannosidases in vivo, eitheralone or in conjunction with other N-glycan modifying enzymes, efficienttrimming of high mannose structures to Man₃GlcNAc₂ is obtained on hostglycoproteins.

[0556] In a preferred embodiment, the Sf9 mannosidase III (Genbankgi:2245567 (D. Jarvis, et al. Glycobiology 1997 7:113-127)) is clonedinto a yeast integration plasmid under the control of a constitutive orinducible promoter (see Example 26). The amount of Class III mannosidaseactivity is optimized while restricting adverse effects on the cell.This involves altering promoter strength and may include using aninducible or otherwise regulatable promoter to better control theexpression of these proteins.

[0557] In addition to expressing the wild-type Class III mannosidase,modified forms of the Class III mannosidase can be expressed to enhancecellular localization and activity. This is achieved through thecombinatorial DNA library approach of the invention by fusing varyinglengths of the catalytic domain of Class III mannosidase(s) toendogenous yeast targeting regions, as described herein.

[0558] Class III Mannosidase Hydrolysis of Glycosidic Linkages

[0559] The method of the present invention also encompasses themechanism in which the catalytically active domain of Class III enzymeshydrolyzes the Manα1,3 and/or Manα1,6 and/or Manα1,2 glycosidic linkageson an oligosaccharide e.g. Man₅GlcNAc₂ or Man₈GlcNAc₂ structures toproduce Man₃GlcNAc₂, a desired intermediate for further N-glycanprocessing in a lower eukaryote.

[0560] In a first embodiment, the hydrolysis of the glycosidic linkagesoccurs sequentially. The enzyme hydrolyzes at least one glycosidiclinkage and conformationally rotates to hydrolyze the other glycosidiclinkages.

[0561] In a second embodiment, the hydrolysis ofthe Manα1,6 and Manα1,3glycosidic linkages occurs simultaneously. In another embodiment, theenzyme specifically hydrolyzes Manα1,2 glycosidic linkages. Theintermediate produced is a substrate for further Golgi processingwherein other glycosylation enzymes such asN-acetylglucosaminyltransferases (GnTs), galactosyltransferases (GaITs)and sialyltransferases (STs) can subsequently modify it to produce adesired glycoform. FIG. 36C illustrates the oligosaccharideintermediates (e.g. Man₄GlcNAc₂, Man₃GlcNAc₂) produced via themannosidase III pathway.

[0562] Host Cells of the Invention

[0563] A preferred host cell of the invention is a lower eukaryoticcell, e.g., yeast, a unicellular and multicellular or filamentousfungus. However, a wide variety of host cells are envisioned as beinguseful in the methods of the invention. Plant cells or insect cells, forinstance, may be engineered to express a human-like glycoproteinaccording to the invention. Likewise, a variety of non-human, mammalianhost cells may be altered to express more human-like or otherwisealtered glycoproteins using the methods of the invention. As one ofskill in the art will appreciate, any eukaryotic host cell (including ahuman cell) may be used in conjunction with a library of the inventionto express one or more chimeric proteins which is targeted to asubcellular location, e.g., organelle, in the host cell where theactivity of the protein is modified, and preferably is enhanced. Such aprotein is preferably—but need not necessarily be—an enzyme involved inprotein glycosylation, as exemplified herein. It is envisioned that anyprotein coding sequence may be targeted and selected for modifiedactivity in a eukaryotic host cell using the methods described herein.

[0564] Lower eukaryotes that are able to produce glycoproteins havingthe attached N-glycan Man₅GlcNAc₂ are particularly useful because (a)lacking a high degree of mannosylation (e.g. greater than 8 mannoses perN-glycan, or especially 30-40 mannoses), they show reducedimmunogenicity in humans; and (b) the N-glycan is a substrate forfurther glycosylation reactions to formn an even more human-likeglycoform, e.g., by the action of GlcNAc transferase I (FIG. 1B; β1,2GnTI) to form GlcNAcMan₅GlcNAc₂. A yield is obtained of greater than 30mole %, more preferably a yield of 50-100 mole %, glycoproteins withN-glycans having a Man₅GlcNAc₂ structure. In a preferred embodiment,more than 50% of the Man₅GlcNAc₂ structure is shown to be a substratefor a GnTI activity and can serve as such a substrate in vivo.

[0565] Preferred lower eukaryotes of the invention include but are notlimited to: Pichia pastoris, Pichia finlandica, Pichia trehalophila,Pichia koclamae, Pichia membranaefaciens, Pichia opuntiae, Pichiathermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi,Pichia stiptis, Pichia methanolica, Pichia sp., Saccharomycescerevisiae, Saccharomyces sp., Hansenula polymorpha, Kluyveromyces sp.,Kluyveromyces lactis, Candida albicans, Aspergillus nidulans,Aspergillus niger, Aspergillus oryzae, Trichoderma reseei, Chrysosporiumlucknowense, Fusarium sp. Fusarium gramineum, Fusarium venenalum andNeurospora crassa.

[0566] In each above embodiment, the method is directed to making a hostcell in which the oligosaccharide precursors are enriched inMan₅GlcNAc₂. These structures are desirable because they may then beprocessed by treatment in vitro, for example, using the method of Marasand Contreras, U.S. Pat. No. 5,834,251. In a preferred embodiment,however, precursors enriched in Man₅GlcNAc₂ are processed by at leastone further glycosylation reaction in vivo—with glycosidases (e.g.,α-mannosidases) and glycosyltransferases (e.g., GnTI)—to producehuman-like N-glycans. Oligosaccharide precursors enriched inMan₅GlcNAc₂, for example, are preferably processed to those havingGlcNAcMan_(X)GlcNAc₂ core structures, wherein X is 3, 4 or 5, and ispreferably 3. N-glycans having a GlcNAcMan_(X)GlcNAc₂ core structurewhere X is greater than 3 may be converted to GlcNAcMan₃GlcNAc₂, e.g.,by treatment with an α-1,3 and/or α-1,6 mannosidase activity, whereapplicable. Additional processing of GlcNAcMan₃GlcNAc₂ by treatment withglycosyltransferases (e.g., GnTII) produces GlcNAc₂Man₃GlcNAc₂ corestructures which may then be modified, as desired, e.g., by ex vivotreatment or by heterologous expression in the host cell of additionalglycosylation enzymes, including glycosyltransferases, sugartransporters and mannosidases (see below), to become human-likeN-glycans.

[0567] Preferred human-like glycoproteins which may be producedaccording to the invention include those which comprise N-glycans havingseven or fewer, or three or fewer, mannose residues; and which compriseone or more sugars selected from the group consisting of galactose,GlcNAc, sialic acid, and fucose.

[0568] While lower eukaryotic host cells are preferred, a wide varietyof host cells having the aforementioned properties are envisioned asbeing useful in the methods of the invention. Plant cells, for instance,may be engineered to express a human-like glycoprotein according to theinvention. Likewise, a variety of non-human, mammalian host cells may bealtered to express more human-like glycoproteins using the methods ofthe invention. An appropriate host cell can be engineered, or one of themany such mutants already described in yeasts may be used. A preferredhost cell of the invention, as exemplified herein, is ahypermannosylation-minus (OCH1) mutant in Pichia pastoris.

[0569] Formation of Complex N-glycans

[0570] Formation of complex N-glycan synthesis is a sequential processby which specific sugar residues are removed and attached to the coreoligosaccharide structure. In higher eukaryotes, this is achieved byhaving the substrate sequentially exposed to various processing enzymes.These enzymes carry out specific reactions depending on their particularlocation within the entire processing cascade. This “assembly line”consists of ER, early, medial and late Golgi, and the trans Golginetwork all with their specific processing environment. To re-create theprocessing of human glycoproteins in the Golgi and ER of lowereukaryotes, numerous enzymes (e.g. glycosyltransferases, glycosidases,phosphatases and transporters) have to be expressed and specificallytargeted to these organelles, and preferably, in a location so that theyfunction most efficiently in relation to their environment as well as toother enzymes in the pathway.

[0571] Because one goal of the methods described herein is to achieve arobust protein production strain that is able to perform well in anindustrial fermentation process, the integration of multiple genes intothe host cell chromosome involves careful planning. As described above,one or more genes which encode enzymes known to be characteristic ofnon-human glycosylation reactions are preferably deleted. The engineeredcell strain is transformed with a range of different genes encodingdesired activities, and these genes are transformed in a stable fashion,thereby ensuring that the desired activity is maintained throughout thefermentation process.

[0572] Any combination of the following enzyme activities may beengineered singly or multiply into the host using methods of theinvention: sialyltransferases, mannosidases, fucosyltransferases,galactosyltransferases, GlcNAc transferases, ER and Golgi specifictransporters (e.g. syn- and antiport transporters for UDP-galactose andother precursors), other enzymes involved in the processing ofoligosaccharides, and enzymes involved in the synthesis of activatedoligosaccharide precursors such as UDP-galactose andCMP-N-acetylneuraminic acid. Preferably, enzyme activities areintroduced on one or more nucleic acid molecules (see also below).Nucleic acid molecules may be introduced singly or multiply, e.g., inthe context of a nucleic acid library such as a combinatorial library ofthe invention. It is to be understood, however, that single or multipleenzymatic activities may be introduced into a host cell in any fashion,including but not limited to protein delivery methods and/or by use ofone or more nucleic acid molecules without necessarily using a nucleicacid library or combinatorial library of the invention.

[0573] Expression of Glycosyltransferases to Produce Complex N-glycans:

[0574] With DNA sequence information, the skilled artisan can clone DNAmolecules encoding GnT activities (e.g., Example 3). Using standardtechniques well-known to those of skill in the art, nucleic acidmolecules encoding GnTI, II, III, IV or V (or encoding catalyticallyactive fragments thereof) may be inserted into appropriate expressionvectors under the transcriptional control of promoters and otherexpression control sequences capable of driving transcription in aselected host cell of the invention, e.g., a fungal host such as Pichiasp., Kluyveromyces sp. and Aspergillus sp., as described herein, suchthat one or more of these mammalian GnT enzymes may be activelyexpressed in a host cell of choice for production of a humanlike complexglycoprotein (e.g., Examples 8, 15, 17, 19.).

[0575] Several individual glycosyltransferases have been cloned andexpressed in S.cerevisiae (GalT, GnTI), Aspergillus nidulans (GnTI) andother fungi, without however demonstrating the desired outcome of“humanization” on the glycosylation pattern of the organisms (Yoshida etal. (1999) Glycobiology 9(1):53-8; Kalsner et al. (1995) Glycoconj. J.12(3):360-370). It was speculated that the carbohydrate structurerequired to accept sugars by the action of such glycosyltransferases wasnot present in sufficient amounts, which most likely contributed to thelack of complex N-glycan formation.

[0576] A preferred method of the invention provides the functionalexpression of a glycosyltransferase, such as GnTI, GnTII and GnTIII (orother GnTs such as GnTIV and GnTVI and combinations of any of the above)in the early, medial or late Golgi apparatus, as well as ensuring asufficient supply of UDP-GlcNAc (e.g., by expression of a UDP-GlcNActransporter; see below).

[0577] Methods for Providing Sugar Nucleotide Precursors to the GolgiApparatus:

[0578] For a glycosyltransferase to function satisfactorily in theGolgi, the enzyme requires a sufficient concentration of an appropriatenucleotide sugar, which is the high-energy donor of the sugar moietyadded to a nascent glycoprotein. In humans, the full range of nucleotidesugar precursors (e.g. UDP-N-acetylglucosamine,UDP-N-acetylgalactosamine, CMP-N-acetylneuraminic acid, UDP-galactose,etc.) are generally synthesized in the cytosol and transported into theGolgi, where they are attached to the core oligosaccharide byglycosyltransferases.

[0579] To replicate this process in non-human host cells such as lowereukaryotes, sugar nucleoside specific transporters have to be expressedin the Golgi to ensure adequate levels of nucleoside sugar precursors(Sommers and Hirschberg (1981) J. Cell Biol. 91(2):A406-A406; Sommersand Hirschberg (1982) J. Biol. Chem. 257(18):811-817; Perez andHirschberg (1987) Methods in Enzymology 138:709-715). Nucleotide sugarsmay be provided to the appropriate compartments, e.g., by expressing inthe host microorganism an exogenous gene encoding a sugar nucleotidetransporter. The choice of transporter enzyme is influenced by thenature of the exogenous glycosyltransferase being used. For example, aGlcNAc transferase may require a UDP-GlcNAc transporter, afucosyltransferase may require a GDP-fucose transporter, agalactosyltransferase may require a UDP-galactose transporter, and asialyltransferase may require a CMP-sialic acid transporter.

[0580] The added transporter protein conveys a nucleotide sugar from thecytosol into the Golgi apparatus, where the nucleotide sugar may bereacted by the glycosyltransferase, e.g. to elongate an N-glycan. Thereaction liberates a nucleoside diphosphate or monophosphate, e.g. UDP,GDP, or CMP. Nucleoside monophosphates can be directly exported from theGolgi in exchange for nucleoside triphosphate sugars by an antiportmechanism. Accumulation of a nucleoside diphosphate, however, inhibitsthe further activity of a glycosyltransferase. As this reaction appearsto be important for efficient glycosylation, it is frequently desirableto provide an expressed copy of a gene encoding a nucleotidediphosphatase. The diphosphatase (specific for UDP or GDP asappropriate) hydrolyzes the diphosphonucleoside to yield a nucleosidemonosphosphate and inorganic phosphate.

[0581] Suitable transporter enzymes, which are typically of mammalianorigin, are described below. Such enzymes may be engineered into aselected host cell using the methods of the invention.

[0582] In another example, α2,3- or α2,6-sialyltransferase capsgalactose residues with sialic acid in the trans-Golgi and TGN of humansleading to a mature form of the glycoprotein (FIG. 1B). To reengineerthis processing step into a metabolically engineered yeast or funguswill require (1) α 2,3- or α 2,6-sialyltransferase activity and (2) asufficient supply of CMP-N-acetyl neuraminic acid, in the late Golgi ofyeast. To obtain sufficient α 2,3-sialyltransferase activity in the lateGolgi, for example, the catalytic domain of a known sialyltransferase(e.g. from humans) has to be directed to the late Golgi in fungi (seeabove). Likewise, transporters have to be engineered to allow thetransport of CMP-N-acetyl neuraminic acid into the late Golgi. There iscurrently no indication that fungi synthesize or can even transportsufficient amounts of CMP-N-acetyl neuraminic acid into the Golgi.Consequently, to ensure the adequate supply of substrate for thecorresponding glycosyltransferases, one has to metabolically engineerthe production of CMP-sialic acid into the fungus.

[0583] UDP-N-acetylglucosamine

[0584] The cDNA of human UDP-N-acetylglucosamine transporter, which wasrecognized through a homology search in the expressed sequence tagsdatabase (dbEST), has been cloned (Ishida, 1999 J. Biochem. 126(1):68-77). The mammalian Golgi membrane transporter forUDP-N-acetylglucosamine was cloned by phenotypic correction with cDNAfrom canine kidney cells (MDCK) of a recently characterizedKluyveromyces lactis mutant deficient in Golgi transport of the abovenucleotide sugar (Guillen et al. (1998) Proc. Natl. Acad. Sci. USA95(14):7888-7892). Results demonstrate that the mammalian GolgiUDP-GlcNAc transporter gene has all of the necessary information for theprotein to be expressed and targeted functionally to the Golgi apparatusof yeast and that two proteins with very different amino acid sequencesmay transport the same solute within the same Golgi membrane (Guillen etal. (1998) Proc. Natl. Acad. Sci. USA 95(14):7888-7892).

[0585] Accordingly, one may incorporate the expression of a UDP-GlcNActransporter in a host cell by means of a nucleic acid construct whichmay contain, for example: (1) a region by which the transformedconstruct is maintained in the cell (e.g. origin of replication or aregion that mediates chromosomal integration), (2) a marker gene thatallows for the selection of cells that have been transformed, includingcounterselectable and recyclable markers such as ura3 or T-urf13(Soderholm et al. (2001) Biolechniques 31 (2):306-10) or other wellcharacterized selection-markers (e.g., his4, bla, Sh ble etc.), (3) agene or fragment thereof encoding a functional UDP-GlcNAc transporter(e.g. from K.lactis, (Abeijon, (1996) Proc. Natl. Acad. Sci. U.S.A.93:5963-5968), or from H.sapiens (Ishida et al. (1996) J. Biochem.(Tokyo) 120(6):1074-8), and (4) a promoter activating the expression ofthe above mentioned localization/catalytic domain fusion constructlibrary. Example 8 shows the addition of a Kluyveromyces lactis MNN2-2gene (Genbank AN AF106080) encoding the UDP-GlcNAc transporter in a P.pastoris PBP-3. FIG. 10A and 10B compares the MALDI-TOF N-glycanprofiles of a P. pastoris strain without the UDP-GlcNAc transporter anda P. pastoris strain with the UDP-GlcNAc transporter (PBP-3),respectively. The P. pastoris PBP-3 exhibits a single prominent peak at1457 (m/z) consistent with its identification as GlcNAcMan₅GlcNAc₂ [b].

[0586] GDP-Fucose

[0587] The rat liver Golgi membrane GDP-fucose transporter has beenidentified and purified by Puglielli, L. and C. B. Hirschberg(Puglielli, 1999 J. Biol. Chem. 274(50):35596-35600). The correspondinggene has not been identified, however, N-terminal sequencing can be usedfor the design of oligonucleotide probes specific for the correspondinggene. These oligonucleotides can be used as probes to clone the geneencoding for GDP-fucose transporter.

[0588] UDP-Galaclose

[0589] Two heterologous genes, gmal2(+) encoding alpha1,2-galactosyltransferase (alpha 1,2 GalT) from Schizosaccharomycespombe and (hUGT2) encoding human UDP-galactose (UDP-Gal) transporter,have been functionally expressed in S.cerevisiae to examine theintracellular conditions required for galactosylation. Correlationbetween protein galactosylation and UDP-galactose transport activityindicated that an exogenous supply of UDP-Gal transporter, rather thanalpha 1,2 GalT played a key role for efficient galactosylation inS.cerevisiae (Kainuma, 1999 Glycobiology 9(2): 133-141). Likewise, anUDP-galactose transporter from S. pombe was cloned (Segawa, 1999 FebsLetters 451(3): 295-298).

[0590] CMP-N-acetylneuraminic Acid (CMP-Sialic Acid).

[0591] Human CMP-sialic acid transporter (hCST) has been cloned andexpressed in Lec 8 CHO cells (Aoki et al. (1999) J. Biochem. (Tokyo)126(5):940-50; Eckhardt et al. (1997) Eur. J. Biochem. 248(1):187-92).The functional expression of the murine CMP-sialic acid transporter wasachieved in Saccharomyces cerevisiae (Berninsone et al. (1997) J. Biol.Chem. 272(19):12616-9). Sialic acid has been found in some fungi,however it is not clear whether the chosen host system will be able tosupply sufficient levels of CMP-Sialic acid. Sialic acid can be eithersupplied in the medium or alternatively fungal pathways involved insialic acid synthesis can also be integrated into the host genome.

[0592] Expression of Diphosphatases:

[0593] When sugars are transferred onto a glycoprotein, either anucleoside diphosphate or monophosphate is released from the sugarnucleotide precursors. While monophosphates can be directly exported inexchange for nucleoside triphosphate sugars by an antiport mechanism,diphosphonucleosides (e.g. GDP) have to be cleaved by phosphatases (e.g.GDPase) to yield nucleoside monophosphates and inorganic phosphate priorto being exported. This reaction appears to be important for efficientglycosylation, as GDPase from S.cerevisiae has been found to benecessary for mannosylation. However, the enzyme only has 10% of theactivity towards UDP (Berninsone et al. (1994) J. Biol. Chem.269(1):207-211). Lower eukaryotes often do not have UDP-specificdiphosphatase activity in the Golgi as they do not utilize UDP-sugarprecursors for glycoprotein synthesis in the Golgi. Schizosaccharomycespombe, a yeast which adds galactose residues to cell wallpolysaccharides (from UDP-galactose), was found to have specific UDPaseactivity, further suggesting the requirement for such an enzyme(Berninsone et al. (1994) J. Biol. Chem. 269(1):207-211). UDP is knownto be a potent inhibitor of glycosyltransferases and the removal of thisglycosylation side product is important to prevent glycosyltransferaseinhibition in the lumen of the Golgi.

[0594] Recombinant Vectors

[0595] A variety of expression vectors may be used to express thenucleotide sequences of the present invention (see, e.g., Example 13).The sequences may be operatively linked to an expression controlsequence in a suitable vector for transformation of a host cell. In oneembodiment, a sequence of the present invention is operably linked to avector designated pJN348, which comprises a GAPDH promoter, a NotI AscIPacI restriction site cassette, Cycll transcriptional terminator, theura3 selection cassette for expression in a P. pasioris YSH-1 (Amp^(r)).

[0596] In a preferred embodiment, the vector comprises a catalyticallyactive fragment of a mannosidase II enzyme as set forth in the abovedescription. Other suitable expression vectors for use in yeast andfilamentous fungi are well-known in the art.

[0597] Methods for Altering N-Glycans in a Host by Expressing a TargetedEnzymatic Activity from a Nucleic Acid Molecule

[0598] The present invention further provides a method for producing ahuman-like glycoprotein in a non-human host cell comprising the step ofintroducing into the cell one or more nucleic acid molecules whichencode an enzyme or enzymes for production of the Man₅GlcNAc₂carbohydrate structure. In one preferred embodiment, a nucleic acidmolecule encoding one or more mannosidase activities involved in theproduction of Man₅GlcNAc₂ from Man₈GlcNAc₂ or Man₉GlcNAc₂ is introducedinto the host. The invention additionally relates to methods for makingaltered glycoproteins in a host cell comprising the step of introducinginto the host cell a nucleic acid molecule which encodes one or moreglycosylation enzymes or activities. Preferred enzyme activities areselected from the group consisting of UDP-GlcNAc transferase,UDP-galactosyltransferase, GDP-fucosyltransferase,CMP-sialyltransferase, UDP-GlcNAc transporter, UDP-galactosetransporter, GDP-fucose transporter, CMP-sialic acid transporter, andnucleotide diphosphatases. In a particularly preferned embodiment, thehost is selected or engineered to express two or more enzymaticactivities in which the product of one activity increases substratelevels of another activity, e.g., a glycosyltransferase and acorresponding sugar transporter, e.g., GnTI and UDP-GlcNAc transporteractivities. In another preferred embodiment, the host is selected orengineered to expresses an activity to remove products which may inhibitsubsequent glycosylation reactions, e.g. a UDP- or GDP-specificdiphosphatase activity.

[0599] Preferred methods of the invention involve expressing one or moreenzymatic activities from a nucleic acid molecule in a host cell andcomprise the step of targeting at least one enzymatic activity to adesired subcellular location (e.g., an organelle) by forming a fusionprotein comprising a catalytic domain of the enzyme and a cellulartargeting signal peptide, e.g., a heterologous signal peptide which isnot normally ligated to or associated with the catalytic domain. Thefusion protein is encoded by at least one genetic construct (“fusionconstruct”) comprising a nucleic acid fragment encoding a cellulartargeting signal peptide ligated in the same translational reading frame(“in-frame”) to a nucleic acid fragment encoding an enzyme (e.g.,glycosylation enzyme), or catalytically active fragment thereof.

[0600] The targeting signal peptide component of the fusion construct orprotein is preferably derived from a member of the group consisting of:membrane-bound proteins of the ER or Golgi, retrieval signals, Type IImembrane proteins, Type I membrane proteins, membrane spanningnucleotide sugar transporters, mannosidases, sialyltransferases,glucosidases, mannosyltransferases and phosphomannosyltransferases.

[0601] The catalytic domain component of the fusion construct or proteinis preferably derived from a glycosidase, mannosidase or aglycosyltransferase activity derived from a member of the groupconsisting of GnTI, GnTII, GnTIII, GnTIV, GnTV, GnTVI, GaIT,Fucosyltransferase and Sialyltransferase. The catalytic domainpreferably has a pH optimum within 1.4 pH units of the average pHoptimum of other representative enzymes in the organelle in which theenzyme is localized, or has optimal activity at a pH between 5.1 and8.0. In a preferred embodiment, the catalytic domain encodes amannosidase selected from the group consisting of C. elegans mannosidaseIA, C. elegans mannosidase IB, D. melanogaster mannosidase IA, H.sapiens mannosidase IB, P. citrinum mannosidase I, mouse mannosidase IA,mouse mannosidase IB, A. nidulans mannosidase IA, A. nidulansmannosidase IB, A. nidulans mannosidase IC, mouse mannosidase II, C.elegans mannosidase II, H. sapiens mannosidase II, and mannosidase III.

[0602] Selecting a Glycosylation Enzyme: pH Optima and SubcellularLocalization

[0603] In one embodiment of the invention, a human-like glycoprotein ismade efficiently in a non-human eukaryotic host cell by introducing intoa subcellular compartment of the cell a glycosylation enzyme selected tohave a pH optimum similar to the pH optima of other enzymes in thetargeted subcellular compartment. For example, most enzymes that areactive in the ER and Golgi apparatus of S.cerevisiae have pH optima thatare between about 6.5 and 7.5 (see Table 3). Because the glycosylationof proteins is a highly evolved and efficient process, the internal pHof the ER and the Golgi is likely also in the range of about 6-8. Allprevious approaches to reduce mannosylation by the action of recombinantmannosidases in fungal hosts, however, have introduced enzymes that havea pH optimum of around pH 5.0 (Martinet et al. (1998) Biotech. Letters20(12): 1171-1177, and Chiba et al. (1998) J. Biol. Chem. 273(41):26298-26304). At pH 7.0, the in vitro determined activity of thosemannosidases is reduced to less than 10%, which is likely insufficientactivity at their point of use, namely, the ER and early Golgi, for theefficient in vivo production of Man₅GlcNAc₂ on N-glycans.

[0604] Accordingly, a preferred embodiment of this invention targets aselected glycosylation enzyme (or catalytic domain thereof), e.g., anα-mannosidase, to a subcellular location in the host cell (e.g., anorganelle) where the pH optimum of the enzyme or domain is within 1.4 pHunits of the average pH optimum of other representative marker enzymeslocalized in the same organelle(s). The pH optimum of the enzyme to betargeted to a specific organelle should be matched with the pH optimumof other enzymes found in the same organelle to maximize the activityper unit enzyme obtained. Table 3 summarizes the activity ofmannosidases from various sources and their respective pH optima. Table4 summarizes their typical subcellular locations. TABLE 3 Mannosidasesand their pH optimum. pH Source Enzyme optimum Reference Aspergillussaitoi α-1,2-mannosidase 5.0 Ichishima et al., 1999 Biochem. J. 339(Pt3): 589-597 Trichoderma reesei α-1,2-mannosidase 5.0 Maras et al., 2000J. Biotechnol. 77(2-3): 255-263 Penicillium citrinum α-D-1,2-mannosidase5.0 Yoshida et al., 1993 Biochem. J. 290(Pt 2): 349-354 C. elegansα-1,2-mannosidase 5.5 FIG. 11 herein Aspergillus nidulansα-1,2-mannosidase 6.0 Eades and Hintz, 2000 Gene 255(1): 25-34 Homosapiens α-1,2-mannosidase 6.0 IA(Golgi) Homo sapiens IBα-1,2-mannosidase 6.0 (Golgi) Lepidopteran insect Type 1 α-1,2-Man₆- 6.0Ren et al., 1995 Biochem. cells mannosidase 34(8): 2489-2495 Homosapiens α-D-mannosidase 6.0 Chandrasekaran et al., 1984 Cancer Res.44(9): 4059-68 Xanthomonas α-1,2,3-mannosidase 6.0 U.S. Pat. No.6,300,113 manihotis Drosophila α-1,2-mannosidase 6.2 Reported hereinmelanogaster Mouse IB (Golgi) α-1,2-mannosidase 6.5 Schneikert andHerscovics, 1994 Glycobiology. 4(4): 445-50 Bacillus sp. (secreted)α-D-1,2-mannosidase 7.0 Maruyama et al., 1994 Carbohydrate Res. 251:89-98

[0605] In a preferred embodiment, a particular enzyme or catalyticdomain is targeted to a subcellular location in the host cell by meansof a chimeric fusion construct encoding a protein comprising a cellulartargeting signal peptide not normally associated with the enzymaticdomain. Preferably, an enzyme or domain is targeted to the ER, theearly, medial or late Golgi, or the trans Golgi apparatus of the hostcell.

[0606] In a more preferred embodiment, the targeted glycosylation enzymeis a mannosidase, glycosyltransferase or a glycosidase. In an especiallypreferred embodiment, mannosidase activity is targeted to the ER or cisGolgi, where the early reactions of glycosylation occur. While thismethod is useful for producing a human-like glycoprotein in a non-humanhost cell, it will be appreciated that the method is also useful moregenerally for modifying carbohydrate profiles of a glycoprotein in anyeukaryotic host cell, including human host cells.

[0607] Targeting sequences which mediate retention of proteins incertain organelles of the host cell secretory pathway are well-known anddescribed in the scientific literature and public databases, asdiscussed in more detail below with respect to libraries for selectionof targeting sequences and targeted enzymes. Such subcellular targetingsequences may be used alone or in combination to target a selectedglycosylation enzyme (or catalytic domain thereof) to a particularsubcellular location in a host cell, i.e., especially to one where theenzyme will have enhanced or optimal activity based on pH optima or thepresence of other stimulatory factors.

[0608] When one attempts to trim high mannose structures to yieldMan₅GlcNAc₂ in the ER or the Golgi apparatus of a host cell such asS.cerevisiae, for example, one may choose any enzyme or combination ofenzymes that (1) has a sufficiently close pH optimum (i.e. between pH5.2 and pH 7.8), and (2) is known to generate, alone or in concert, thespecific isomeric Man₅GlcNAc₂ structure required to accept subsequentaddition of GlcNAc by GnTI. Any enzyme or combination of enzymes that isshown to generate a structure that can be converted to GlcNAcMan₅GlcNAc₂by GnTI in vitro would constitute an appropriate choice. This knowledgemay be obtained from the scientific literature or experimentally.

[0609] For example, one may determine whether a potential mannosidase.can convert Man₈GlcNAc₂-2AB (2-aminobenzamide) to Man₅GlcNAc₂-AB andthen verify that the obtained Man₅GlcNAc₂-2AB structure can serve asubstrate for GnTI and UDP-GlcNAc to give GlcNAcMan₅GlcNAc₂ in vitro.Mannosidase IA from a human or murine source, for example, would be anappropriate choice (see, e.g., Example 4). Examples described hereinutilize 2-aminobenzamide labeled N-linked oligomannose followed by HPLCanalysis to make this determination. TABLE 4 Cellular location and pHoptima of various glycosylation-related enzymes of S. cerevisiae. pHGene Activity Location optimum Reference(s) KTR1 α-1,2 Golgi 7.0 Romeroet al. (1997) mannosyl- Biochem. J. 321(Pt 2): transferase 289-295 MNS1α-1,2- ER 6.5 mannosidase CWH41 glucosidase I ER 6.8 — mannosyl- Golgi7-8 Lehele and Tanner transferase (1974) Biochim. Biophys. Acta 350(1):225-235 KRE2 α-1,2 Golgi 6.5-9.0 Romero et al. (1997) mannosyl- Biochem.J. 321(Pt 2): transferase 289-295

[0610] Accordingly, a glycosylation enzyme such as an α-1,2-mannosidaseenzyme used according to the invention has an optimal activity at a pHof between 5.1 and 8.0. In a preferred embodiment, the enzyme has anoptimal activity at a pH of between 5.5 and 7.5. The C. elegansmannosidase enzyme, for example, works well in the methods of theinvention and has an apparent pH optimum of about 5.5). Preferredmannosidases include those listed in Table 3 having appropriate pHoptima, e.g. Aspergillus nidulans, Homo sapiens IA (Golgi), Homo sapiensIB (Golgi), Lepidopteran insect cells (IPLB-SF21AE), Homo sapiens, mouseIB (Golgi), Xanthomonas manihotis, Drosophila melanogaster and C.elegans.

[0611] The experiment which illustrates the pH optimum for anα-1,2-mannosidase enzyme is described in Example 7. A chimeric fusionprotein BB27-2 (Saccharomyces MNN10 (s)/C. elegans mannosidase IB Δ31),which leaks into the medium was subjected to various pH ranges todetermine the optimal activity of the enzyme. The results of theexperiment show that the α-1,2-mannosidase has an optimal pH of about5.5 for its function (FIG. 11).

[0612] In a preferred embodiment, a single cloned mannosidase gene isexpressed in the host organism. However, in some cases it may bedesirable to express several different mannosidase genes, or severalcopies of one particular gene, in order to achieve adequate productionof Man₅GlcNAc₂. In cases where multiple genes are used, the encodedmannosidases preferably all have pH optima within the preferred range ofabout 5.1 to about 8.0, or especially between about 5.5 and about 7.5.Preferred mannosidase activities include α-1,2-mannosidases derived frommouse, human, Lepidoptera, Aspergillus nidulans, or Bacillus sp.,C.elegans, D.melanogaster, P.citrinum, X.laevis or A.nidulans.

[0613] In Vivo Alteration of Host Cell Glycosylation Using aCombinatorial DNA Library

[0614] Certain methods of the invention are preferably (but need notnecessarily be) carried out using one or more nucleic acid libraries. Anexemplary feature of a combinatorial nucleic acid library of theinvention is that it comprises sequences encoding cellular targetingsignal peptides and sequences encoding proteins to be targeted (e.g.,enzymes or catalytic domains thereof, including but not limited to thosewhich mediate glycosylation).

[0615] In one embodiment, a combinatorial nucleic acid librarycomprises: (a) at least two nucleic acid sequences encoding differentcellular targeting signal peptides; and (b) at least one nucleic acidsequence encoding a polypeptide to be targeted. In another embodiment, acombinatorial nucleic acid library comprises: (a) at least one nucleicacid sequence encoding a cellular targeting signal peptide; and (b) atleast two nucleic acid sequences encoding a polypeptide to be targetedinto a host cell. As described further below, a nucleic acid sequencederived from (a) and a nucleic acid sequence derived from (b) areligated to produce one or more fusion constructs encoding a cellulartargeting signal peptide functionally linked to a polypeptide domain ofinterest. One example of a functional linkage is when the cellulartargeting signal peptide is ligated to the polypeptide domain ofinterest in the same translational reading frame (“in-frame”).

[0616] In a preferred embodiment, a combinatorial DNA library expressesone or more fusion proteins comprising cellular targeting signalpeptides ligated in-frame to catalytic enzyme domains. The encodedfusion protein preferably comprises a catalytic domain of an enzymeinvolved in mammalian- or human-like modification of N-glycans. In amore preferred embodiment, the catalytic domain is derived from anenzyme selected from the group consisting of mannosidases,glycosyltransferases and other glycosidases which is ligated in-frame toone or more targeting signal peptides. The enzyme domain may beexogenous and/or endogenous to the host cell. A particularly preferredsignal peptide is one normally associated with a protein that undergoesER to Golgi transport.

[0617] The combinatorial DNA library of the present invention may beused for producing and localizing in vivo enzymes involved in mammalian-or human-like N-glycan modification. The fusion constructs of thecombinatorial DNA library are engineered so that the encoded enzymes arelocalized in the ER, Golgi or the trans-Golgi network of the host cellwhere they are involved in producing particular N-glycans on aglycoprotein of interest. Localization of N-glycan modifying enzymes ofthe present invention is achieved through an anchoring mechanism orthrough protein-protein interaction where the localization peptideconstructed from the combinatorial DNA library localizes to a desiredorganelle of the secretory pathway such as the ER, Golgi or the transGolgi network.

[0618] An example of a useful N-glycan, which is produced efficientlyand in sufficient quantities for further modification by human-like(complex) glycosylation reactions is Man₅GlcNAc₂. A sufficient amount ofMan₅GlcNAc₂ is needed on a glycoprotein of interest for furtherhuman-like processing in vivo (e.g., more than 30 mole %). TheMan₅GlcNAc₂ intermediate may be used as a substrate for further N-glycanmodification to produce GlcNAcMan₅GlcNAc₂ (FIG. 1B; see above).Accordingly, the combinatorial DNA library of the present invention maybe used to produce enzymes which subsequently produce GlcNAcMan₅GlcNAc₂,or other desired complex N-glycans, in a useful quantity.

[0619] A further aspect of the fusion constructs produced using thecombinatorial DNA library of the present invention is that they enablesufficient and often near complete intracellular N-glycan trimmingactivity in the engineered host cell. Preferred fusion constructsproduced by the combinatorial DNA library of the invention encode aglycosylation enzyme, e.g., a mannosidase, which is effectivelylocalized to an intracellular host cell compartment and thereby exhibitsvery little and preferably no extracellular activity. The preferredfusion constructs of the present invention that encode a mannosidaseenzyme are shown to localize where the N-glycans are modified, namely,the ER and the Golgi. The fusion enzymes of the present invention aretargeted to such particular organelles in the secretory pathway wherethey localize and act upon N-glycans such as Man₈GlcNAc₂ to produceMan₅GlcNAc₂ on a glycoprotein of interest.

[0620] Enzymes produced by the combinatorial DNA library of the presentinvention can modify N-glycans on a glycoprotein of interest as shownfor K3 or IFN-β proteins expressed in P.pastoris, as shown in FIGS. 5and 6, respectively (see also Examples 2 and 4). It is, however,appreciated that other types of glycoproteins, without limitation,including erythropoietin, cytokines such as interferon-α, interferon-β,interferon-γ, interferon-ω, and granulocyte-CSF, coagulation factorssuch as factor VIII, factor IX, and human protein C, soluble IgEreceptor α-chain, IgG, IgG fragments, IgM, urokinase, chymase, and ureatrypsin inhibitor, IGF-binding protein, epidermal growth factor, growthhormone-releasing factor, annexin V fusion protein, angiostatin,vascular endothelial growth factor-2, myeloid progenitor inhibitoryfactor-1, osteoprotegerin, α-1 antitrypsin, DNase II and α-feto proteinsmay be glycosylated in this way.

[0621] Constructing a Combinatorial DNA Library of Fusion Constructs:

[0622] A combinatorial DNA library of fusion constructs features one ormore cellular targeting signal peptides (“targeting peptides”) generallyderived from N-terminal domains of native proteins (e.g., by makingC-terminal deletions). Some targeting peptides, however, are derivedfrom the C-terminus of native proteins (e.g. SEC12). Membrane-boundproteins of the ER or the Golgi are preferably used as a source fortargeting peptide sequences. These proteins have sequences encoding acytosolic tail (ct), a transmembrane domain (tmd) and a stem region (sr)which are varied in length. These regions are recognizable by proteinsequence alignments and comparisons with known homologs and/or otherlocalized proteins (e.g., comparing hydrophobicity plots).

[0623] The targeting peptides are indicated herein as short (s), medium(m) and long (l) relative to the parts of a type II membrane. Thetargeting peptide sequence indicated as short (s) corresponds to thetransmembrane domain (tmd) of the membrane-bound protein. The targetingpeptide sequence indicated as long (l) corresponds to the length of thetransmembrane domain (tmd) and the stem region (sr). The targetingpeptide sequence indicated as medium (m) corresponds to thetransmembrane domain (tmd) and approximately half the length of the stemregion (sr). The catalytic domain regions are indicated herein by thenumber of nucleotide deletion with respect to its wild-typeglycosylation enzyme.

[0624] Sub-libraries

[0625] In some cases a combinatorial nucleic acid library of theinvention may be assembled directly from existing or wild-type genes. Ina preferred embodiment, the DNA library is assembled from the fusion oftwo or more sub-libraries. By the in-frame ligation of thesub-libraries, it is possible to create a large number of novel geneticconstructs encoding useful targeted protein domains such as those whichhave glycosylation activities.

[0626] Catalytic Domain Sub-Libbraries Encoding Glycosylation Activities

[0627] One useful sub-library includes DNA sequences encoding enzymessuch as glycosidases (e.g., mannosidases), glycosyltransferases (e.g.,fucosyl-transferases, galactosyltransferases, glucosyltransferases),GlcNAc transferases and sialyltransferases. Catalytic domains may beselected from the host to be engineered, as well as from other relatedor unrelated organisms. Mammalian, plant, insect, reptile, algal orfungal enzymes are all useful and should be chosen to represent a broadspectrum of biochemical properties with respect to temperature and pHoptima. In a preferred embodiment, genes are truncated to give fragmentssome of which encode the catalytic domains of the enzymes. By removingendogenous targeting sequences, the enzymes may then be redirected andexpressed in other cellular loci.

[0628] The choice of such catalytic domains may be guided by theknowledge of the particular environment in which the catalytic domain issubsequently to be active. For example, if a particular glycosylationenzyme is to be active in the late Golgi, and all known enzymes of thehost organism in the late Golgi have a certain pH optimum, or the lateGolgi is known to have a particular pH, then a catalytic domain ischosen which exhibits adequate, and preferably maximum, activity at thatpH, as discussed above.

[0629] Targeting Peptide Sequence Sub-Libraries

[0630] Another useful sub-library includes nucleic acid sequencesencoding targeting signal peptides that result in localization of aprotein to a particular location within the ER, Golgi, or trans Golginetwork. These targeting peptides may be selected from the host organismto be engineered as well as from other related or unrelated organisms.Generally such sequences fall into three categories: (1) N-terminalsequences encoding a cytosolic tail (ct), a transmembrane domain (tmd)and part or all of a stem region (sr), which together or individuallyanchor proteins to the inner (lumenal) membrane of the Golgi; (2)retrieval signals which are generally found at the C-terminus such asthe HDEL or KDEL tetrapeptide; and (3) membrane spanning regions fromvarious proteins, e.g., nucleotide sugar transporters, which are knownto localize in the Golgi.

[0631] In the first case, where the targeting peptide consists ofvarious elements (ct, tmd and sr), the library is designed such that thect, the tmd and various parts of the stem region are represented.Accordingly, a preferred embodiment of the sub-library of targetingpeptide sequences includes ct, tmd, and/or sr sequences frommembrane-bound proteins of the ER or Golgi. In some cases it may bedesirable to provide the sub-library with varying lengths of srsequence. This may be accomplished by PCR using primers that bind to the5′ end of the DNA encoding the cytosolic region and employing a seriesof opposing primers that bind to various parts of the stem region.

[0632] Still other useful sources of targeting peptide sequences includeretrieval signal peptides, e.g. the tetrapeptides HDEL or KDEL, whichare typically found at the C-terminus of proteins that are transportedretrograde into the ER or Golgi. Still other sources of targetingpeptide sequences include (a) type II membrane proteins, (b) the enzymeslisted in Table 3, (c) membrane spanning nucleotide sugar transportersthat are localized in the Golgi, and (d) sequences referenced in TABLE 5Sources of useful compartmental targeting sequences Gene or Location ofGene Sequence Organism Function Product MNS1 A. nidulansα-1,2-mannosidase ER MNS1 A. niger α-1,2-mannosidase ER MNS1 S.cerevisiae α-1,2-mannosidase ER GLS1 S. cerevisiae glucosidase ER GLS1A. niger glucosidase ER GLS1 A. nidulans glucosidase ER HDEL Universalin fungi retrieval signal ER at C-terminus SEC12 S. cerevisiae COPIIvesicle protein ER/Golgi SEC12 A. niger COPII vesicle protein ER/GolgiOCH1 S. cerevisiae 1,6-mannosyltransferase Golgi (cis) OCH1 P. pastoris1,6-mannosyltransferase Golgi (cis) MNN9 S. cerevisiae1,6-mannosyltransferase Golgi complex MNN9 A. niger undetermined GolgiVAN1 S. cerevisiae undetermined Golgi VAN1 A. niger undetermined GolgiANP1 S. cerevisiae undetermined Golgi HOC1 S. cerevisiae undeterminedGolgi MNN10 S. cerevisiae undetermined Golgi MNN10 A. niger undeterminedGolgi MNN11 S. cerevisiae undetermined Golgi (cis) MNN11 A. nigerundetermined Golgi (cis) MNT1 S. cerevisiae 1,2-mannosyltransferaseGolgi (cis, medial KTR1 P. pastoris undetermined Golgi (medial) KRE2 P.pastoris undetermined Golgi (medial) KTR3 P. pastoris undetermined Golgi(medial) MNN2 S. cerevisiae 1,2-mannosyltransferase Golgi (medial) KTR1S. cerevisiae undetermined Golgi (medial) KTR2 S. cerevisiaeundetermined Golgi (medial) MNN1 S. cerevisiae 1,3-mannosyltransferaseGolgi (trans) MNN6 S. cerevisiae Phosphomannosyltransferase Golgi(trans) 2,6 ST H. sapiens 2,6-sialyltransferase trans Golgi networkUDP-Gal T S. pombe UDP-Gal transporter Golgi

[0633] In any case, it is highly preferred that targeting peptidesequences are selected which are appropriate for the particularenzymatic activity or activities to function optimally within thesequence of desired glycosylation reactions. For example, in developinga modified host microorganism capable of terminal sialylation of nascentN-glycans, a process which occurs in the late Golgi in humans, it isdesirable to utilize a sub-library of targeting peptide sequencesderived from late Golgi proteins. Similarly, the trimming of Man₈GlcNAc₂by an α-1,2-mannosidase to give Man₅GlcNAc₂ is an early step in complexN-glycan formation in humans (FIG. 1B). It is therefore desirable tohave this reaction occur in the ER or early Golgi of an engineered hostmicroorganism. A sub-library encoding ER and early Golgi retentionsignals is used.

[0634] A series of fusion protein constructs (i.e., a combinatorial DNAlibrary) is then constructed by functionally linking one or a series oftargeting peptide sequences to one or a series of sequences encodingcatalytic domains. In a preferred embodiment, this is accomplished bythe in-frame ligation of a sub-library comprising DNA encoding targetingpeptide sequences (above) with a sub-library comprising DNA encodingglycosylation enzymes or catalytically active fragments thereof (seebelow).

[0635] The resulting library comprises synthetic genes encodingtargeting peptide sequence-containing fusion proteins. In some cases itis desirable to provide a targeting peptide sequence at the N-terminusof a fusion protein, or in other cases at the C-terminus. In some cases,targeting peptide sequences may be inserted within the open readingframe of an enzyme, provided the protein structure of individual foldeddomains is not disrupted. Each type of fusion protein is constructed (ina step-wise directed or semi-random fashion) and optimal constructs maybe selected upon transformation of host cells and characterization ofglycosylation patterns in transformed cells using methods of theinvention.

[0636] Generating Additional Sequence Diversity

[0637] The method of this embodiment is most effective when a nucleicacid, e.g., a DNA library transformed into the host contains a largediversity of sequences, thereby increasing the probability that at leastone transformant will exhibit the desired phenotype. Single amino acidmutations, for example, may drastically alter the activity ofglycoprotein processing enzymes (Romero et al. (2000) J. Biol. Chem.275(15):11071-4). Accordingly, prior to transformation, a DNA library ora constituent sub-library may be subjected to one or more techniques togenerate additional sequence diversity. For example, one or more roundsof gene shuffling, error prone PCR, in vitro mutagenesis or othermethods for generating sequence diversity, may be performed to obtain alarger diversity of sequences within the pool of fusion constructs.

[0638] Expression Control Sequences

[0639] In addition to the open reading frame sequences described above,it is generally preferable to provide each library construct withexpression control sequences, such as promoters, transcriptionterminators, enhancers, ribosome binding sites, and other functionalsequences as may be necessary to ensure effective transcription andtranslation of the fusion proteins upon transformation of fusionconstructs into the host organism.

[0640] Suitable vector components, e.g., selectable markers, expressioncontrol sequences (e.g., promoter, enhancers, terminators and the like)and, optionally, sequences required for autonomous replication in a hostcell, are selected as a function of which particular host cell ischosen. Selection criteria for suitable. vector components for use in aparticular mammalian or a lower eukaryotic host cell are routine.Preferred lower eukaryotic host cells of the invention include Pichiapastoris, Pichiafinlandica, Pichia trehalophila, Pichia koclamae, Pichiamembranaefaciens, Pichia opuntiae, Pichia thermotolerans, Pichiasalictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichiamethanolica, Pichia sp., Saccharomyces cerevisiae, Saccharomyces sp.,Hansenula polymorpha, Kluyveromyces sp., Kluyveromyces lactis, Candidaalbicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oiyzae,Trichoderma reesei, Chrysosporium lucknowense, Fusarium sp. Fusariumgramilleum, Fusarium venenatum and Neurospora crassa. Where the host isPichia pastoris, suitable promoters include, for example, the AOX1,AOX2, GAPDH and P40 promoters.

[0641] Selectable Markers

[0642] It is also preferable to provide each construct with at least oneselectable marker, such as a gene to impart drug resistance or tocomplement a host metabolic lesion. The presence of the marker is usefulin the subsequent selection of transformants; for example, in yeast theURA3, HIS4, SUC2. G418, BLA, or SH BLE genes may be used. A multitude ofselectable markers are known and available for use in yeast, fungi,plant, insect, mammalian and other eukaryotic host cells.

[0643] Transformation

[0644] The nucleic acid library is then transformed into the hostorganism. In yeast, any convenient method of DNA transfer may be used,such as electroporation, the lithium chloride method, or the spheroplastmethod. In filamentous fungi and plant cells, conventional methodsinclude particle bombardment, electroporation and agrobacterium mediatedtransformation. To produce a stable strain suitable for high-densityculture (e.g., fermentation in yeast), it is desirable to integrate theDNA library constructs into the host chromosome. In a preferredembodiment, integration occurs via homologous recombination, usingtechniques well-known in the art. For example, DNA library elements areprovided with flanking sequences homologous to sequences of the hostorganism. In this manner, integration occurs at a defined site in thehost genome, without disruption of desirable or essential genes.

[0645] In an especially preferred embodiment, library DNA is integratedinto the site of an undesired gene in a host chromosome, effecting thedisruption or deletion of the gene. For example, integration into thesites of the OCH1, MNN1, or MNN4 genes allows the expression of thedesired library DNA while preventing the expression of enzymes involvedin yeast hypermannosylation of glycoproteins. In other embodiments,library DNA may be introduced into the host via a nucleic acid molecule,plasmid, vector (e.g., viral or retroviral vector), chromosome, and maybe introduced as an autonomous nucleic acid molecule or by homologous orrandom integration into the host genome. In any case, it is generallydesirable to include with each library DNA construct at least oneselectable marker gene to allow ready selection of host organisms thathave been stably transformed. Recyclable marker genes such as URA3,which can be selected for or against, are especially suitable.

[0646] Screening and Selection Processes

[0647] After transformation of the host strain with the DNA library,transformants displaying a desired glycosylation phenotype are selected.Selection may be performed in a single step or by a series of phenotypicenrichment and/or depletion steps using any of a variety of assays ordetection methods. Phenotypic characterization may be carried outmanually or using automated high-throughput screening equipment.Commonly, a host microorganism displays protein N-glycans on the cellsurface, where various glycoproteins are localized.

[0648] One may screen for those cells that have the highestconcentration of terminal GlcNAc on the cell surface, for example, orfor those cells which secrete the protein with the highest terminalGlcNAc content. Such a screen may be based on a visual method, like astaining procedure, the ability to bind specific terminal GlcNAc bindingantibodies or lectins conjugated to a marker (such lectins are availablefrom E.Y. Laboratories Inc., San Mateo, Calif.), the reduced ability ofspecific lectins to bind to terminal mannose residues, the ability toincorporate a radioactively labeled sugar in vitro, altered binding todyes or charged surfaces, or may be accomplished by using a FluorescenceAssisted Cell Sorting (FACS) device in conjunction with a fluorophorelabeled lectin or antibody (Guillen et al. (1998) Proc. Natl. Acad. Sci.USA 95(14):7888-7892).

[0649] Accordingly, intact cells may be screened for a desiredglycosylation phenotype by exposing the cells to a lectin or antibodythat binds specifically to the desired N-glycan. A wide variety ofoligosaccharide-specific lectins are available commercially (e.g., fromEY Laboratories, San Mateo, Calif.). Alternatively, antibodies tospecific human or animal N-glycans are available commercially or may beproduced using standard techniques. An appropriate lectin or antibodymay be conjugated to a reporter molecule, such as a chromophore,fluorophore, radioisotope, or an enzyme having a chromogenic substrate(Guillen et al., 1998. Proc. Natl. Acad. Sci. USA 95(14): 7888-7892).

[0650] Screening may then be performed using analytical methods such asspectrophotometry, fluorimetry, fluorescence activated cell sorting, orscintillation counting. In other cases, it may be necessary to analyzeisolated glycoproteins or N-glycans from transformed cells. Proteinisolation may be carried out by techniques known in the art. In apreferred embodiment, a reporter protein is secreted into the medium andpurified by affinity chromatography (e.g. Ni-affinity orglutathione-S-transferase affinity chromatography). In cases where anisolated N-glycan is preferred, an enzyme such asendo-β-N-acetylglucosaminidase (Genzyme Co., Boston, Mass.; New EnglandBiolabs, Beverly, Mass.) may be used to cleave the N-glycans fromglycoproteins. Isolated proteins or N-glycans may then be analyzed byliquid chromatography (e.g. HPLC), mass spectroscopy, or other suitablemeans. U.S. Pat. No. 5,595,900 teaches several methods by which cellswith desired extracellular carbohydrate structures may be identified. Ina preferred embodiment, MALDI-TOF mass spectrometry is used to analyzethe cleaved N-glycans.

[0651] Prior to selection of a desired transformnant, it may bedesirable to deplete the transformed population of cells havingundesired phenotypes. For example, when the method is used to engineer afunctional mannosidase activity into cells, the desired transfomiantswill have lower levels of mannose in cellular glycoprotein. Exposing thetransformed population to a lethal radioisotope of mannose in the mediumdepletes the population of transformants having the undesired phenotype,i.e. high levels of incorporated mannose (Huffaker T C and Robbins P W.,Proc Natl Acad Sci U S A. 1983 December;80(24):7466-70). Alternatively,a cytotoxic lectin or antibody, directed against an undesirableN-glycan, may be used to deplete a transformed population of undesiredphenotypes (e.g., Stanley P and Siminovitch L. Somatic Cell Genet 1977July;3(4):391-405). U.S. Pat. No. 5,595,900 teaches several methods bywhich cells with a desired extracellular carbohydrate structures may beidentified. Repeatedly carrying out this strategy allows for thesequential engineering of more and more complex glycans in lowereukaryotes.

[0652] To detect host cells having on their surface a high degree of thehuman-like N-glycan intermediate GlcNAcMan₃GlcNAc₂, for example, one mayselect for transfonrmants that allow for the most efficient transfer ofGlcNAc by GlcNAc Transferase from UDP-GlcNAc in an in vitro cell assay.This screen may be carried out by growing cells harboring thetransformed library under selective pressure on an agar plate andtransferring individual colonies into a 96-well microtiter plate. Aftergrowing the cells, the cells are centrifuged, the cells resuspended inbuffer, and after addition of UDP-GlcNAc and GnTII, the release of UDPis determined either by HPLC or an enzyme linked assay for UDP.Alternatively, one may use radioactively labeled UDP-GlcNAc and GnTII,wash the cells and then look for the release of radioactive GlcNAc byN-actylglucosaminidase. All this may be carried out manually or may beautomated through the use of high throughput screening equipment.Transformants that release more UDP in the first assay, or moreradioactively labeled GlcNAc in the second assay, are expected to have ahigher degree of GlcNAcMan₃GlcNAc₂ on their surface and thus constitutethe desired phenotype. Similar assays may be adapted to look at theN-glycans on secreted proteins as well.

[0653] Alternatively, one may use any other suitable screen such as alectin binding assay that is able to reveal altered glycosylationpatterns on the surface of transformed cells. In this case the reducedbinding of lectins specific to terminal mannoses may be a suitableselection tool. Galantus nivalis lectin binds specifically to terminalα-1,3 mannose, which is expected to be reduced if sufficient mannosidaseII activity is present in the Golgi. One may also enrich for desiredtransformants by carrying out a chromatographic separation step thatallows for the removal of cells containing a high terminal mannosecontent. This separation step would be carried out with a lectin columnthat specifically binds cells with a high tenrminal mannosecontent:(e.g., Galantus nivalis lectin bound to agarose, Sigma,St.Louis, Mo.) over those that have a low terminal mannose content.

[0654] In addition, one may directly create such fusion proteinconstructs, as additional information on the localization of activecarbohydrate modifying enzymes in different lower eukaryotic hostsbecomes available in the scientific literature. For example, it is knownthat human β1,4-GalTr can be fused to the membrane domain of MNT, amannosyltransferase from S.cerevisiae, and localized to the Golgiapparatus while retaining its catalytic activity (Schwientek et al.(1995) J. Biol. Chem. 270(10):5483-9). If S.cerevisiae or a relatedorganism is the host to be engineered one may directly incorporate suchfindings into the overall strategy to obtain complex N-glycans from sucha host. Several such gene fragments in P.pastoris have been identifiedthat are related to glycosyltransferases in S.cerevisiae and thus couldbe used for that purpose.

[0655] Alteration of Host Cell Glycosylation Using Fusion ConstructsFrom Combinatorial Libraries

[0656] The construction of a preferred combinatorial DNA library isillustrated schematically in FIG. 2 and described in Example 4. Thefusion construct may be operably linked to a multitude of vectors, suchas expression vectors well-known in the art. A wide variety of suchfusion constructs were assembled using representative activities asshown in Table 6. Combinations of targeting peptide/catalytic domainsmay be assembled for use in targeting mannosidase, glycosyltransferaseand glycosidase activities in the ER, Golgi and the trans Golgi networkaccording to the invention. Surprisingly, the same catalytic domain mayhave no effect to a very profound effect on N-glycosylation patterns,depending on the type of targeting peptide used (see, e.g., Table 7,Example 4).

[0657] Mannosidase I Fusion Constructs

[0658] A representative example of a mannosidase fusion constructderived from a combinatorial DNA library of the invention is pFB8, whichhas a truncated Saccharomyces SEC12(m) targeting peptide (988-1296nucleotides of SEC12 from SwissProt P11655) ligated in-frame to a 187N-terminal amino acid deletion of a mouse α-mannosidase IA (Genbank AN6678787). The nomenclature used herein, thus, refers to the targetingpeptide/catalytic domain region of a glycosylation enzyme asSaccharomyces SEC12 (m)/mouse mannosidase IA Δ187. The encoded fusionprotein localizes in the ER by means of the SEC12 targeting peptidesequence while retaining its mannosidase catalytic domain activity andis capable of producing in vivo N-glycans having a Man₅GlcNAc₂ structure(Example 4; FIGS. 6F and 7B).

[0659] The fusion construct pGC5, Saccharomyces MNS1(m)/mousemannosidase IB Δ99, is another example of a fusion construct havingintracellular mannosidase trimming activity (Example 4; FIGS. 5D and8B). Fusion construct pBC18-5 (Saccharomyces VAN1(s)/C. elegansmannosidase IB Δ80) is yet another example of an efficient fusionconstruct capable of producing in vivo N-glycans having a Man₅GlcNAc₂structure. By creating a combinatorial DNA library of these and othersuch mannosidase fusion constructs according to the invention, a skilledartisan may distinguish and select those constructs having optimalintracellular trimming activity from those having relatively low or noactivity. Methods using combinatorial DNA libraries of the invention areadvantageous because only a select few mannosidase fusion constructs mayproduce a particularly desired N-glycan in vivo.

[0660] In addition, mannosidase trimming activity may be specific to aparticular protein of interest. Thus, it is to be further understoodthat not all targeting peptide/mannosidase catalytic domain fusionconstructs may function equally well to produce the proper glycosylationon a glycoprotein of interest. Accordingly, a protein of interest may beintroduced into a host cell transfected with a combinatorial DNA libraryto identify one or more fusion constructs which express a mannosidaseactivity optimal for the protein of interest. One skilled in the artwill be able to produce and select optimal fusion construct(s) using thecombinatorial DNA library approach described herein.

[0661] It is apparent, moreover, that other such fusion constructsexhibiting localized active mannosidase catalytic domains (or moregenerally, domains of any enzyme) may be made using techniques such asthose exemplified in Example 4 and described herein. It will be a matterof routine experimentation for one skilled in the art to make and usethe combinatorial DNA library of the present invention to optimize, forexample, Man₅GlcNAc₂ production from a library of fusion constructs in aparticular expression vector introduced into a particular host cell.

[0662] Glycosyltransferase Fusion Constructs

[0663] Similarly, a glycosyltransferase combinatorial DNA library wasmade using the methods of the invention. A combinatorial DNA library ofsequences derived from glycosyltransferase I (GnTI) activities wereassembled with targeting peptides and screened for efficient productionin a lower eukaryotic host cell of a GlcNAcMan₅GlcNAc₂ N-glycanstructure on a marker glycoprotein. A fusion construct shown to produceGlcNAcMan₅GlcNAc₂ (pPB104), Saccharomyces MNN9(s)/human GnTI Δ38 wasidentified (Example 8). A wide variety of such GnTI fusion constructswere assembled (Example 8, Table 10). Other combinations of targetingpeptide/GnTI catalytic domains can readily be assembled by making acombinatorial DNA library. It is also apparent to one skilled in the artthat other such fusion constructs exhibiting glycosyltransferaseactivity may be made as demonstrated in Example 8. It will be a matterof routine experimentation for one skilled in the art to use thecombinatorial DNA library method described herein to optimizeGlcNAcMan₅GlcNAc₂ production using a selected fusion construct in aparticular expression vector and host cell line.

[0664] As stated above for mannosidase fusion constructs, not alltargeting peptide/GnTI catalytic domain fusion constructs will functionequally well to produce the proper glycosylation on a glycoprotein ofinterest as described herein. However, one skilled in the art will beable to produce and select optimal fusion construct(s) using a DNAlibrary approach as described herein. Example 8 illustrates a preferredembodiment of a combinatorial DNA library comprising targeting peptidesand GnTI catalytic domain fusion constructs involved in producingglycoproteins with predominantly GlcNAcMan₅GlcNAc₂ structure.

[0665] Using Multiple Fusion Constructs to Alter Host Cell Glycosylation

[0666] In another example of using the methods and libraries of theinvention to alter host cell glycosylation, a P.pastoris strain with anOCH1 deletion that expresses a reporter protein (K3) was transformedwith multiple fusion constructs isolated from combinatorial libraries ofthe invention to convert high mannose N-glycans to human-like N-glycans(Example 8). First, the mannosidase fusion construct pFB8 (SaccharomycesSEC12 (m)/mouse mannosidase IA Δ187) was transformed into a P.pastorisstrain lacking 1,6 initiating mannosyltransferases activity (i.e. och1deletion; Example 1). Second, pPB103 comprising a K.lactis MNN2-2 gene(Genbank AN AF106080) encoding an UDP-GlcNAc transporter was constructedto increase further production of GlcNAcMan₅GlcNAc₂. The addition of theUDP-GlcNAc transporter increased production of GlcNAcMan₅GlcNAc₂significantly in the P.pastoris strain as illustrated in FIG. 10B.Third, pPB104 comprising Saccharomyces MNN9 (s)/human GnTI Δ38 wasintroduced into the strain. This P.pastoris strain is referred to as“PBP-3.”

[0667] It is understood by one skilled in the art that host cells suchas the above-described yeast strains can be sequentially transfornnedand/or co-transforned with one or more expression vectors. It is alsounderstood that the order of transformation is not particularly relevantin producing the glycoprotein of interest. The skilled artisanrecognizes the routine modifications of the procedures disclosed hereinmay provide improved results in the production of the glycoprotein ofinterest.

[0668] The importance of using a particular targeting peptide sequencewith a particular catalytic domain sequence becomes readily apparentfrom the experiments described herein. The combinatorial DNA libraryprovides a tool for constructing enzyme fusions that are involved inmodifying N-glycans on a glycoprotein of interest, which is especiallyuseful in producing human-like glycoproteins. (Any enzyme fusion,however, may be selected using libraries and methods of the invention.)Desired transfonnants expressing appropriately targeted, activeα-1,2-mannosidase produce K3 with N-glycans of the structure Man₅GlcNAc₂as shown in FIGS. 5D and 5E. This confers a reduced molecular mass tothe cleaved glycan compared to the K3 of the parent OCH1 deletionstrain, as was detected by MALDI-TOF mass spectrometry in FIG. 5C.

[0669] Similarly, the same approach was used to produce another secretedglycoprotein: IFN-β comprising predominantly Man₅GlcNAc₂. TheMan₅GlcNAc₂ was removed by PNGase digestion (Papac et al. 1998Glycobiology 8, 445-454) and subjected to MALDI-TOF as shown in FIGS.6A-6F. A single prominent peak at 1254 (m/z) confirms Man₅GlcNA₂production on IFN-β in FIGS. 6E (pGC5) (Saccharomyces MNS1(m)/mousemannosidase IBΔ99) and 6F (pFB8) (Saccharomyces SEC12 (m)/mousemannosidase IA Δ187). Furthermore, in the P.pastoris strain PBP-3comprising pFB8 (Saccharomyces SEC12 (m)/niouse mannosidase IA Δ187),pPB104 (Saccharomyces MNN9 (s)/human GnTI Δ38) and pPB103 (K.lactisMNN2-2 gene), the hybrid N-glycan GlcNAcMan₅GlcNAc₂ [b] was detected byMALDI-TOF (FIG. 10).

[0670] After identifying transformants with a high degree of mannosetrimming, additional experiments were performed to confirm thatmannosidase (trimming) activity occurred in vivo and was notpredominantly the result of extracellular activity in the growth medium(Example 6; FIGS. 7-9).

[0671] Golgi α-Mannosidase II Fusion Constructs

[0672] As provided by the methods of the invention, a combinatorial DNAlibrary of Golgi α-mannosidase II was made by fusing the catalyticdomain of several mannosidase II enzymes to an array of cellulartargeting peptide signals (Example 14). The resulting more than 500combinatorial fusion constructs were introduced into a P. pastorisstrain capable of producing the human precursor of complexglycosylation, GlcNAcMan₅GlcNAc₂ YSH-1 (Example 17) on the reporter K3.Only a small subset of strains about (<5%) were capable ofquantitatively converting GlcNAcMan₅GlcNAc₂ to GlcNAcMan₃GlcNAc₂. Thesestrains were isolated and subsequently transformed with a combinatoriallibrary of several hundred GnTII/leader peptide fusions. Screening forthe presence of GlcNAc₂Man₃GlcNAc₂ allowed for the isolation of strainsthat were able to secrete homogeneous complex glycan, as exemplified bystrain YSII-44 (Example 19).

[0673] A representative example of a Golgi α-mannosidase II fusionconstruct derived from a combinatorial DNA library of the invention ispKD53, which a truncated S.cerevisiae MNN2(s) targeting peptide (1-108nucleotides of MNN2 from SwissProt P38069) ligated in-frame to a 74N-terninal amino acid deletion of a D.melanogaster golgi α-mannosidaseII (Genbank AN X77652). The nomenclature used herein, thus, refers tothe targeting peptide/catalytic domain region of a glycosylation enzymeas S. cerevisiae MNN2(s)/D.melanogaster mannosidase II Δ74. The encodedfusion protein localizes in the Golgi by means of the MNN2(s) targetingpeptide sequence while retaining its mannosidase catalytic domainactivity and is capable of producing in vivo N-glycans having apredominant GlcNAcMan₃GlcNAc₂ structure (Example 18).

[0674] Another example of a Golgi α-mannosidase II fusion constructderived from a combinatorial DNA library of the invention is pKD1, whicha truncated Saccharonyces GLS1(s) targeting peptide (1-102 nucleotidesof GLS1 from SwissProt P53008) ligated in-frame to a 74 N-terminal aminoacid deletion of a D.melanogaster golgi α-mannosidase II (Genbank ANX77652). The nomenclature used herein, thus, refers to the targetingpeptide/catalytic domain region of a glycosylation enzyme asSaccharomyces GLS1 (s)/D.melanogaster mannosidase II Δ74. The encodedfusion protein localizes in the Golgi by means of the GLS1(s) targetingpeptide sequence while retaining its mannosidase catalytic domainactivity and is capable of producing in vivo N-glycans having apredominant GlcNAcMan₃GlcNAc₂ structure (Example 22).

[0675] Another example of a Golgi α-mannosidase II fusion constructderived from a combinatorial DNA library of the invention is pKD5, whicha truncated Saccharomyces MNS1(m) targeting peptide (1-246 nucleotidesof MNS1 from SwissProt P32906) ligated in-frame to a 74 N-terminal aminoacid deletion of a D.melanogaster golgi α-mannosidase II (Genbank ANX77652). The nomenclature used herein, thus, refers to the targetingpeptide/catalytic domain region of a glycosylation enzyme asSaccharomyces MNS1(m)/D.melanogaster mannosidase II Δ74. The encodedfusion protein localizes in the Golgi by means of the MNS1(m) targetingpeptide sequence while retaining its mannosidase catalytic domainactivity and is capable of producing in vivo N-glycans having aGlcNAcMan₃GlcNAc₂ structure (Example 23). Unlike the uniformnity ofN-glycans present in YSH-27, FIG. 21 shows heterogenous mixture ofN-glycans produced YSH-74. The apparent mediocre trimming activity ofthis mannosidase II enzyme, however, indicates the heterogenity asManα1,2 additions as suggested in FIG. 23, where the GlcNAcMan₃GlcNAc₂peak appears after digestion of YSH-74 with A. saitoiα-1,2-maninosidase. By creating a combinatorial DNA library of these andother such mannosidase fusion constructs according to the invention, askilled artisan may distinguish and select those constructs havingoptimal intracellular trimming activity from those having relatively lowor no activity.

[0676] Methods using combinatorial DNA libraries of the invention areadvantageous because only a select few mannosidase fusion constructs mayproduce a particularly desired N-glycan in vivo.

[0677] In addition, mannosidase trimming activity may be specific to aparticular protein of interest. Thus, it is to be further understoodthat not all targeting peptide/mannosidase catalytic domain fusionconstructs may function equally well to produce the proper glycosylationon a glycoprotein of interest. FIG. 18 shows no apparent activity in aP. pastoris YSH-1 transformed a Golgi α-mannosidase II fusion constructderived from a combinatorial DNA library of the invention pKD16, which atruncated Saccharomyces MNN9(m) targeting peptide (1-273 nucleotides ofMNN9 from SwissProt P39107) ligated in-frame to a 74 N-terminal aminoacid deletion of a D.melanogaster golgi α-mannosidase II (Genbank ANX77652). Accordingly, a protein of interest may be introduced into ahost cell transformed with a combinatorial DNA library to identify oneor more fusion constructs which express a mannosidase activity optimalfor the protein of interest.

[0678] One skilled in the art will be able to produce and select optimalfusion construct(s) using the combinatorial DNA library approachdescribed herein.

[0679] Host Cells

[0680] Although the present invention is exemplified using a P.pastorishost organism, it is to be understood by those skilled in the art thatother eukaryotic host cells, including other species of yeast and fungalhosts, may be altered as described herein to produce human-likeglycoproteins. The techniques described herein for identification anddisruption of undesirable host cell glycosylation genes, e.g. OCH1, isunderstood to be applicable for these and/or other homologous orfunctionally related genes in other eukaryotic host cells such as otheryeast and fungal strains. As described in Example 9, och1 mnn1 geneswere deleted from K.lactis to engineer a host cell leading to N-glycansthat are completely converted to Man₅GlcNAc₂ by 1,2-mannosidase (FIG.12C).

[0681] The MNN1 gene was cloned from K.lactis as described in Example 9.

[0682] The nucleic acid and deduced amino acid sequences of the K.lactisMNN1 gene are shown in SEQ ID NOS: 16 and 17, respectively. Usinggene-specific primers, a construct was made to delete the MNN1 gene fromthe genome of K.lactis (Example 9). Host cells depleted in och1 and mnn1activities produce N-glycans having a Man₉GlcNAc₂ carbohydrate structure(see, e.g., FIG. 10). Such host cells may be engineered further using,e.g., methods and libraries of the invention, to produce mammalian- orhuman-like glycoproteins.

[0683] Thus, in another embodiment, the invention provides an isolatednucleic acid molecule having a nucleic acid sequence comprising orconsisting of at least forty-five, preferably at least 50, morepreferably at least 60 and most preferably 75 or more nucleotideresidues of the K.lactis MNN1 gene (SEQ ID NO: 16), and homologs,variants and derivatives thereof. The invention also provides nucleicacid molecules that hybridize under stringent conditions to theabove-described nucleic acid molecules. Similarly, isolated polypeptides(including muteins, allelic variants, fragments, derivatives, andanalogs) encoded by the nucleic acid molecules of the invention areprovided. In addition, also provided are vectors, including expressionvectors, which comprise a nucleic acid molecule of the invention, asdescribed further herein. Similarly host cells transformed with thenucleic acid molecules or vectors of the invention are provided.

[0684] Another aspect of the present invention thus relates to anon-human eukaryotic host strain expressing glycoproteins comprisingmodified N-glycans that resemble those made by human-cells. Performingthe methods of the invention in species other than yeast and fungalcells is thus contemplated and encompassed by this invention. It iscontemplated that a combninatorial nucleic acid library of the presentinvention may be used to select constructs that modify the glycosylationpathway in any eukaryotic host cell system. For example, thecombinatorial libraries of the invention may also be used in plants,algae and insects, and in other eukaryotic host cells, includingmammalian and human cells, to localize proteins, including glycosylationenzymes or catalytic domains thereof, in a desired location along a hostcell secretory pathway. Preferably, glycosylation enzymes or catalyticdomains and the like are targeted to a subcellular location along thehost cell secretory pathway where they are capable of functioning, andpreferably, where they are designed or selected to function mostefficiently.

[0685] Preferred host cells of the present invention include Pichiapastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae,Pichia niembranaefaciens, Pichia opuntiae, Pichia thermotolerans, Pichiasalictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichiamethanolica, Pichia sp., Saccharomyces cerevisiae, Saccharomyces sp.,Hansenula polymorpha, Kluyveromyces sp., Kluyveromyces lactis, Candidaalbicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae,Trichoderma reesei, Chrysosporium lucknowense, Fusarium sp., Fusariumgramineum, Fusariuum venenatum and Neurospora crassa.

[0686] Plant and insect cells may also be engineered to alter theglycosylation of expressed proteins using the combinatorial library andmethods of the invention. Furthermore, glycosylation in mammalian cells,including human cells, may also be modified using the combinatoriallibrary and methods of the invention. It may be possible, for example,to optimize a particular enzymatic activity or to otherwise modify therelative proportions of various N-glycans made in a mammalian host cellusing the combinatorial library and methods of the invention.

[0687] Examples of modifications to glycosylation which can be affectedusing a method according to this embodiment of the invention are: (1)engineering a eukaryotic host cell to trim mannose residues fromMan₈GlcNAc₂ to yield a Man₅GlcNAc₂ N-glycan; (2) engineering eukaryotichost cell to add an N-acetylglucosamine (GlcNAc) residue to Man₅GlcNAc₂by action of GlcNAc transferase 1; (3) engineering a eukaryotic hostcell to functionally express an enzyme such as an N-acetylglucosaminylTransferase (GnTI, GnTII, GnTIII, GnTIV, GnTV, GnTVI), mannosidase II,fucosyltransferase (FT), galactosyl tranferase (GalT) or asialyltransferase (ST).

[0688] By repeating the method, increasingly complex glycosylationpathways can be engineered into a target host, such as a lowereukaryotic microorganism. In one preferred embodiment, the host organismis transformed two or more times with DNA libraries including sequencesencoding glycosylation activities. Selection of desired phenotypes maybe performed after each round of transformation or alternatively afterseveral transformations have occurred. Complex glycosylation pathwayscan be rapidly engineered in this manner.

[0689] Sequential Glycosylation Reactions

[0690] In a preferred embodiment, such targeting peptide/catalyticdomain libraries are designed to incorporate existing information on thesequential nature of glycosylation reactions in higher eukaryotes.Reactions known to occur early in the course of glycoprotein processingrequire the targeting of enzymes that catalyze such reactions to anearly part of the Golgi or the ER. For example, the trimming ofMan₈GlcNAc₂ to Man₅GlcNAc₂ by mannosidases is an early step in complexN-glycan formation. Because protein processing is initiated in the ERand then proceeds through the early, medial and late Golgi, it isdesirable to have this reaction occur in the ER or early Golgi. Whendesigning a library for mannosidase I localization, for example, onethus attempts to match ER and early Golgi targeting signals with thecatalytic domain of mannosidase I.

[0691] Integration Sites

[0692] As one ultimate goal of this genetic engineering effort is arobust protein production strain that is able to perform well in anindustrial fermentation process, the integration of multiple genes intothe host (e.g., fungal) chromosome preferably involves careful planning.The engineered strain may likely have to be transformed with a range ofdifferent genes, and these genes will have to be transformed in a stablefashion to ensure that the desired activity is maintained throughout thefermentation process. As described herein, any combination of variousdesired enzyme activities may be engineered into the fungal proteinexpression host, e.g., sialyltransferases, mannosidases,fucosyltransferases, galactosyltransferases, glucosyltransferases,GlcNAc transferases, ER and Golgi specific transporters (e.g. syn andantiport transporters for UDP-galactose and other precursors), otherenzymes involved in the processing of oligosaccharides, and enzymesinvolved in the synthesis of activated oligosaecharide precursors suchas UDP-galactose, CMP-N-acetylneuraminic acid. Examples of preferredmethods for modifying glycosylation in a lower eukaryotic host cell,such as Pichia pastoris, are shown in Table 6. TABLE 6 Some preferredembodiments for modifying glycosylation in a lower eukaroyticmicroorganism Suitable Suitable Suitable Sources of SuitableTransporters Desired Catalytic Localization Gene and/or StructureActivities Sequences Deletions Phosphatases Man₅GlcNAc₂ α-1,2- Mns1(N-terminus, OCH1 none mannosidase S. cerevisiae) MNN4 (murine, Och1(N-terminus, MNN6 human, S. cerevisiae, Bacillus sp., P. pastoris) A.nidulans) Ktr1 Mnn9 Mnt1 (S. cerevisiae) KDEL, HDEL (C-terminus)GlcNAcMan₅GlcNAc₂ GlcNAc Och1 (N-terminus, OCH1 UDP-GlcNAc Transferase1, S. cerevisiae, MNN4 transporter (human, P. pastoris) MNN6 (human,murine, murine, rat KTR1 (N-terminus) K. lactis) etc.) Mnn1 (N-terminus,UDPase (human) S. cerevisiae) Mnt1 (N-terminus, S. cerevisiae) GDPase(N-terminus, S. cerevisiae) GlcNAcMan₃GlcNAc₂ mannosidase II Ktr1 OCH1UDP-GlcNAc Mnn1 (N-terminus, MNN4 transporter S. cerevisiae) MNN6(human, murine, Mnt1 (N-terminus, K. lactis) S. cerevisiae) UDPase(human) Kre2/Mnt1 (S. cerevisiae) Kre2 (P. pastoris) Ktr1 (S.cerevisiae) Ktr1 (P. pastoris) Mnn1 (S. cerevisiae)GlcNAc₍₂₋₄₎Man₃GlcNAc₂ GlcNAc Mnn1 (N-terminus, OCH1 UDP-GlcNAcTransferase II, S. cerevisiae) MNN4 transporter III, IV, V Mnt1(N-terminus, MNN6 (human, murine, (human, S. cerevisiae) K. lactis)murine) Kre2/Mnt1 UDPase (human) (S. cerevisiae) Kre2 (P. pastoris) Ktr1(S. cerevisiae) Ktr1 (P. pastoris) Mnn1 (S. cerevisiae)Gal₍₁₋₄₎GlcNAc₍₂₋₄₎− β-1,4- Mnn1 (N-terminus, OCH1 UDP-GalactoseMan₃GlcNAc₂ Galactosyl S. cerevisiae) MNN4 transporter transferaseMnt1(N-terminus, MNN6 (human, S. pombe) (human) S. cerevisiae) Kre2/Mnt1(S. cerevisiae) Kre2 (P. pastoris) Ktr1 (S. cerevisiae) Ktr1 (P.pastoris) Mnn1 (S. cerevisiae) NANA₍₁₋₄₎− α-2,6- KTR1 OCH1 CMP-Sialicacid Gal₍₁₋₄₎GlcNAc₍₂₋₄₎− Sialyltransferase MNN1 (N-terminus, MNN4transporter Man₃GlcNAc₂ (human) S. cerevisiae) MNN6 (human) α-2,3- MNT1(N-terminus, Sialyltransferase S. cerevisiae) Kre2/Mnt1 (S. cerevisiae)Kre2 (P. pastoris) Ktr1 (S. cerevisiae) Ktr1 (P. pastoris) MNN1 (S.cerevisiae)

[0693] As any strategy to engineer the formation of complex N-glycansinto a host cell such as a lower eukaryote involves both the eliminationas well as the addition of particular glycosyltransferase activities, acomprehensive scheme will attempt to coordinate both requirements. Genesthat encode enzymes that are undesirable serve as potential integrationsites for genes that are desirable. For example, 1,6 mannosyltransferaseactivity is a hallmark of glycosylation in many known lower eukaryotes.The gene encoding alpha-α1,6 mannosyltransferase (OCH1) has been clonedfrom S.cerevisiae and mutations in the gene give rise to a viablephenotype with reduced mannosylation. The gene locus encoding alpha-α1,6mannosyltransferase activity therefore is a prime target for theintegration of genes encoding glycosyltransferase activity. In a similarmanner, one can choose a range of other chromosomal integration sitesthat, based on a gene disruption event in that locus, are expected to:(1) improve the cell's ability to glycosylate in a more human-likefashion, (2) improve the cell's ability to secrete proteins, (3) reduceproteolysis of foreign proteins and (4) improve other characteristics ofthe process that facilitate purification or the fermentation processitself.

[0694] Target Glycoproteins

[0695] The methods described herein are useful for producingglycoproteins, especially glycoproteins used therapeutically in humans.Glycoproteins having specific glycoforms may be especially useful, forexample, in the targeting of therapeutic proteins. For example,mannose-6-phosphate has been shown to direct proteins to the lysosome,which may be essential for the proper function of several enzymesrelated to lysosomal storage disorders such as Gaucher's, Hunter's,Hurler's, Scheie's, Fabry's and Tay-Sachs disease, to mention just afew. Likewise, the addition of one or more sialic acid residues to aglycan side chain may increase the lifetime of a therapeuticglycoprotein in vivo after administration. Accordingly, host cells(e.g., lower eukaryotic or mammalian) may be genetically engineered toincrease the extent of terminal sialic acid in glycoproteins expressedin the cells. Alternatively, sialic acid may be conjugated to theprotein of interest in vitro prior to administration using a sialic acidtransferase and an appropriate substrate. Changes in growth mediumcomposition may be employed in addition to the expression of enzymeactivities involved in-human-like glycosylation to produce glycoproteinsmore closely resembling human forms (Weikert et al. (1999) NatureBiotechnology 17, 1116-1121; Werner et al. (1998) Arzneimittelforschung48(8):870-880; Andersen and Goochee (1994) Cur. Opin. Biotechnol.5:546-549; Yang and Butler (2000) Biotechnol.Bioengin. 68(4):370-380).Specific glycan modifications to monoclonal antibodies (e.g. theaddition of a bisecting GlcNAc) have been shown to improve antibodydependent cell cytotoxicity (Umana et al. (1999) Nat. Biotechnol.17(2):176-80), which may be desirable for the production of antibodiesor other therapeutic proteins.

[0696] Therapeutic proteins are typically administered by injection,orally, or by pulmonary or other means. Examples of suitable targetglycoproteins which may be produced according to the invention include,without limitation: erythropoietin, cytokines such as interferon-α,interferon-β, interferon-γ, interferon-ω, and granulocyte-CSF,coagulation factors such as factor VIII, factor IX, and human protein C,soluble IgE receptor α-chain, IgG, IgG fragments, IgM, interleukins,urokinase, chymase, and urea trypsin inhibitor, IGF-binding protein,epidermal growth factor, growth hormone-releasing factor, annexin Vfusion protein, angiostatin, vascular endothelial growth factor-2,myeloid progenitor inhibitory factor-1, osteoprotegerin, α-1-antitrypsinand αfeto proteins.

[0697] Subsequent Glycosyltransferase Activities:N-acetylglucosaminyltransferase II, Galactosyltransferase andSialyltransferase

[0698] In a further aspect of the invention, the newly formed glycansproduced by the Golgi α-mannosidase II enzyme are substrates forsubsequent glycosylation reactions. In one embodiment, GnT II,UDP-GlcNAc and optionally the UDP-GlcNAc transporter cap the newlyformed Manα1,6 branch of the oligosaccharide produced in P. pastorisYSH-37 with a GlcNAc to form GlcNAc₂Man₃GlcNAc₂ (Example 19) In anotherembodiment, other GnTs (e.g. GnT III, GnT IV, GnT V) react upon thetransient GlcNAc₂Man₃GlcNAc₂ substrate. This substrate in turn becomes asubstrate for galactosyltransferases (Example 25) and further processingoccurs with sialyltransferases.

[0699] The following are examples which illustrate the compositions andmethods of this invention. These examples should not be construed aslimiting: the examples are included for the purposes of illustrationonly.

EXAMPLE 1 Cloning and Disruption of the OCH1 Gene in P.pastoris

[0700] A 1215 bp ORF of the P.pastoris OCH1 gene encoding a putativeα-1,6 mannosyltransferase was amplified from P.pastoris genomic DNA(strain X-33, Invitrogen, Carlsbad, Calif.) using the oligonucleotides5′-ATGGCGAAGGCAGATGGCAGT-3′ (SEQ ID NO: 18) and5′-TTAGTCCTTCCAACTTCCTTC-3′ (SEQ ID NO: 19) which were designed based onthe P.pastoris OCH1 sequence (Japanese Patent Application PublicationNo. 8-336387). Subsequently, 2685 bp upstream and 1175 bp downstream ofthe ORF of the OCH1 gene were amplified from a P.pastoris genomic DNAlibrary (Boehm, T. et al. Yeast 1999 May;15(7):563-72) using theinternal oligonucleotides 5′-ACTGCCATCTGCCTTCGCCAT-3′ (SEQ ID NO: 20) inthe OCH1 gene, and 5′-GTAATACGACTCACTATAGGGC-3′ T7 (SEQ ID NO: 21) and5′-AATTAACCCTCACTAAAGGG-3′ T3 (SEQ ID NO: 22) oligonucleotides in thebackbone of the library bearing plasmid lambda ZAP II (Stratagene, LaJolla, Calif.). The resulting 5075 bp fragment was cloned into thepCR2.1-TOPO vector (Invitrogen, Carlsbad, Calif.) and designated pBK9.

[0701] After assembling a gene knockout construct that substituted theOCH1 reading frame with a HIS4 resistance gene, P.pastoris wastransformed and colonies were screened for temperature sensitivity at37° C. OCH1 mutants of S.cerevisiae are temperature sensitive and areslow growers at elevated temperatures. One can thus identify functionalhomologs of OCH1 in P.pastoris by complementing an OCH1 mutant ofS.cerevisiae with a P.pastoris DNA or cDNA library. About 20 temperaturesensitive strains were further subjected to a colony PCR screen toidentify colonies with a deleted och1 gene. Several och1 deletions wereobtained.

[0702] The linearized pBK9.1, which has 2.1 kb upstream sequence and 1.5kb down stream sequence of OCH1 gene cassette carrying Pichia HIS4 gene,was transformed into P.pastoris BK1 [GS115 (his4 Invitrogen Corp., SanDiego, Calif.) carrying the human IFN-β gene in the AOX1 locus] to knockout the wild-type OCH1 gene. The initial screening of transformants wasperformed using histidine drop-out medium followed by replica plating toselect the temperature sensitive colonies. Twenty out of two hundredhistidine-positive colonies showed a temperature sensitive phenotype at37° C. To exclude random integration of pBK9.1 into the Pichia genome,the 20 temperature-sensitive isolates were subjected to colony PCR usingprimers specific to the upstream sequence of the integration site and toHIS4 ORF. Two out of twenty colonies were och1 defective and furtheranalyzed using a Southern blot and a Western blot indicating thefunctional och1 disruption by the och1 knock-out construct. Genomic DNAwere digested using two separate restriction enzymes BglII and ClaI toconfirm the och1 knock-out and to confirm integration at the openreading frame. The Western Blot showed och1 mutants lacking a discreteband produced in the GS115 wild type at 46.2 kDa.

EXAMPLE 2 Engineering of P.pastoris with α-1,2-Mannosidase to ProduceMan₅GlcNAc₂-Containing IFN-β Precursors

[0703] An α-1,2-mannosidase is required for the trimming of Man₈GlcNAc₂to yield Man₅GlcNAc₂, an essential intermediate for complex N-glycanformation. While the production of a Man₅GlcNAc₂ precursor is essential,it is not necessarily sufficient for the production of hybrid andcomplex glycans because the specific isomer of Man₅GlcNAc₂ may or maynot be a substrate for GnTI. An och1 mutant of P.pastoris is engineeredto express secreted human interferon-β under the control of an aoxpromoter. A DNA library is constructed by the in-frame ligation of thecatalytic domain of human mannosidase IB (an α-1,2-mannosidase) with asub-library including sequences encoding early Golgi and ER localizationpeptides. The DNA library is then transformed into the host organism,resulting in a genetically mixed population wherein individualtransformants each express interferon-β as well as a syntheticmannosidase gene from the library. Individual transfonnant colonies arecultured and the production of interferon is induced by addition ofmethanol. Under these conditions, over 90% of the secreted protein isglycosylated interferon-β.

[0704] Supernatants are purified to remove salts and low-molecularweight contaminants by C₁₈ silica reversed-phase chromatography. Desiredtransfonnants expressing appropriately targeted, activeα-1,2-mannosidase produce interferon-β including N-glycans of thestructure Man₅GlcNAc₂, which has a reduced molecular mass compared tothe interferon-β of the parent strain. The purified interferon-β isanalyzed by MALDI-TOF mass spectroscopy and colonies expressing thedesired form of interferon-β are identified.

EXAMPLE 3 Generation of an och1 Mutant Strain Expressing anα-1,2-Mannosidase, GnTI for Production of a Human-Like Glycoprotein.

[0705] The 1215 bp open reading frame of the P.pastoris OCH1 gene aswell as 2685 bp upstream and 1175 bp downstream was amplified by PCR(see also WO 02/00879), cloned into the pCR2.1-TOPO vector (Invitrogen)and designated pBK9. To create an och1 knockout strain containingmultiple, auxotrophic markers, 100 μg of pJN329, a plasmid containing anoch1::URA3 mutant allele flanked with SfiI restriction sites wasdigested with Sfil and used to transform P.pastoris strain JC308(Cereghino et al. Gene 263 (2001) 159-169) by electropqration. Followingincubation on defined medium lacking uracil for 10 days at roomtemperature, 1000 colonies were picked and re-streaked. URA⁺ clones thatwere unable to grow at 37° C., but grew at room temperature, weresubjected to colony PCR to test for the correct integration of theoch1::URA3 mutant allele. One clone that exhibited the expected PCRpattern was designated YJN153. The Kringle 3 domain of human plasminogen(K3) was used as a model protein. A Neo^(R) marked plasmid containingthe K3 gene was transformed into strain YJN153 and a resulting strain,expressing K3, was named BK64-1.

[0706] Plasmid pPB103, containing the Kluyveromyces lactis MNN2-2 genewhich encodes a Golgi UDP-N-acetylglucosamine transporter wasconstructed by cloning a blunt BglII-HindIII fragment from vector pDL02(Abeijon et al. (1996) Proc. Natl. Acad. Sci. U.S.A. 93:5963-5968) intoBglII and BamHI digested and blunt ended pBLADE-SX containing theP.pastoris ADE1 gene (Cereghino et al. (2001) Gene 263:159-169). Thisplasmid was linearized with EcoNI and transformed into strain BK64-1 byelectroporation and one strain confirmed to contain the MNN2-2 by PCRanalysis was named PBP1.

[0707] A library of mannosidase constructs was generated, comprisingin-frame fusions of the leader domains of several type I or type IImembrane proteins from S.cerevisiae and P.pastoris fused with thecatalytic domains of several α-1,2-mannosidase genes from human, mouse,fly, worm and yeast sources (see, e.g., WO02/00879, incorporated hereinby reference). This library was created in a P.pastoris HIS4 integrationvector and screened by linearizing with SalI, transforming byelectroporation into strain PBP1, and analyzing the glycans releasedfrom the K3 reporter protein. One active construct chosen was a chimeraof the 988-1296 nucleotides (C-terminus) of the yeast SEC12 gene fusedwith a N-terminal deletion of the mouse α-1,2-mannosidase IA gene (FIG.3), which was missing the 187 nucleotides. A P.pastoris strainexpressing this construct was named PBP2.

[0708] A library of GnTI constructs was generated, comprising in-framefusions of the same leader library with the catalytic domains of GnTIgenes from human, worm, frog and fly sources (WO 02/00879). This librarywas created in a P.pastoris ARG4 integration vector and screened bylinearizing with AatII, transfonning by electroporation into strainPBP2, and analyzing the glycans released from K3. One active constructchosen was a chimera of the first 120 bp of the S.cerevisiae MNN9 genefuised to a deletion of the human GnTI gene, which was missing the first154 bp. A P.pastoris strain expressing this construct was named PBP3.

EXAMPLE 4 Engineering of P.pastoris to Produce Man₅GlcNAc₂ as thePredominant N-Glycan Structure Using a Combinatorial DNA Library

[0709] An och1 mutant of P.pastoris (see Examples 1 and 3) wasengineered to express and secrete proteins such as the kringle 3 domainof human plasminogen (K3) under the control of the inducible AOXIpromoter. The Kringle 3 domain of human plasminogen (K3) was used as amodel protein. A DNA fragment encoding the K3 was amplified using Pfuturbo polymerase (Strategene, La Jolla, Calif.) and cloned into EcoRIand XbaI sites of pPICZαA (Invitrogen, Carlsbad, Calif.), resulting. ina C-terminal 6-His tag. In order to improve the N-linked glycosylationefficiency of K3 (Hayes et al. 1975 J. Arch. Biochem. Biophys. 171,651-655), Pro₄₆ was replaced with Ser₄₆ using site-directed mutagenesis.The resulting plasmid was designated pBK64. The correct sequence of thePCR construct was confirmed by DNA sequencing.

[0710] A combinatorial DNA library was constructed by the in-frameligation of murine α-1,2-mannosidase IB (Genbank AN 6678787) and IA(Genbank AN 6754619) catalytic domains with a sub-library includingsequences encoding Cop II vesicle, ER, and early Golgi localizationpeptides according to Table 6. The combined DNA library was used togenerate individual fusion constructs, which were then transformed intothe K3 expressing host organism, resulting in a genetically mixedpopulation wherein individual transformants each express K3 as well as alocalization signal/mannosidase fusion gene from the library. Individualtransfomiants were cultured and the production of K3 was induced bytransfer to a methanol containing medium. Under these conditions, after24 hours of induction, over 90% of the protein in the medium was K3. TheK3 reporter protein was purified from the supernatant to remove saltsand low-molecular weight contaminants by Ni-affinity chromatography.Following affinity purification, the protein was desalted by sizeexclusion chromatography on a Sephadex G10 resin (Sigma, St. Louis, Mo.)and either directly subjected to MALDI-TOF analysis described below orthe N-glycans were removed by PNGase digestion as described below(Release of N-glycans) and subjected to MALDI-TOF analysis Miele et al.1997 Biotechnol. Appl. Biochem. 25, 151-157.

[0711] Following this approach, a diverse set of transfonnants wereobtained; some showed no modification of the N-glycans compared to theoch1 knockout strain; and others showed a high degree of mannosetrimming (FIGS. 5D and 5E). Desired transformants expressingappropriately targeted, active α-1,2-mannosidase produced K3 withN-glycans of the structure Man₅GlcNAc₂. This confers a reduced molecularmass to the glycoprotein compared to the K3 of the parent och1 deletionstrain, a difference which was readily detected by MALDI-TOF massspectrometry (FIG. 5). Table 7 indicates the relative Man₅GlcNAc₂production levels. TABLE 7 A representative combinatorial DNA library oflocalization sequences/catalytic domains exhibiting relative levels ofMan₅GlcNAc₂ production. Catalytic Targeting peptide sequences DomainsMNS1(s) MNS1(m) MNS1(l) SEC12(s) SEC12(m) Mouse FB4 FB5 FB6 FB7 FB8mannosidase ++ + − ++ ++++ 1A Δ187 Mouse GB4 GB5 GB6 GB7 GB8 mannosidase++ + + ++ + 1B Δ58 Mouse GC4 GC5 GC6 GC7 GC8 mannosidase − +++ + + + 1BΔ99 Mouse GD4 GD5 GD6 GD7 GD8 mannosidase − − − + + 1B Δ170

[0712] TABLE 8 Another combinatorial DNA library of localizationsequences/catalytic domains exhibiting relative levels of Man₅GlcNAc₂production. Catalytic Targeting peptide sequences Domains VAN1(s)VAN1(m) VAN1(l) MNN10(s) MNN10(m) MNN10(l) C. elegans BC18-5 BC19 BC20BC27 BC28 BC29 mannosidase 1B +++++ ++++ +++ +++++ +++++ +++ Δ80 C.elegans BB18 BB19 BB20 BB18 BB19 BB20 mannosidase 1B +++++ +++++ +++++++++ +++++ ++++ Δ31

[0713] Targeting peptides were selected from MNSI (SwissProt P32906) inS.cerevisiae (long, medium and short) (see supra, Nucleic AcidLibraries; Combinatorial DNA Library of Fusion Constructs) and SEC12(SwissProt P11655) in S.cerevisiae (988-1140 nucleotides: short) and(988-1296: medium). Although the majority of targeting peptide sequenceswere N-terminal deletions, some targeting peptide sequences, such asSEC12, were C-terminal deletions. Catalytic domains used in thisexperiment were selected from mouse mannosidase 1A with a 187 amino acidN-termninal deletion; and mouse mannosidase 1B with a 58, 99 and 170amino acid deletion. The number of (+)s, as used herein, indicates therelative levels of Man₅GlcNAc₂ production. The notation (−) indicates noapparent production of Man₅GlcNAc₂ The notation (+) indicates less than10% production of Man₅GlcNAc₂ The notation (++) indicates about 10-20%production of Man₅GlcNAc₂, The notation with (+++) indicates about20-40% production of Man₅GlcNAc₂. The notation with (++++) indicatesabout 50% production of Man₅GlcNAc₂. The notation with (+++++) indicatesgreater than 50% production of Man₅GlcNAc₂.

[0714] Table 9 shows the relative amounts of Man₅GlcNAc₂ detected on asecreted K3 reporter glycoprotein. Six hundred and eight (608) differentstrains of P.pastoris (Δoch1) were generated by transforming each with asingle construct from a combinatorial genetic library that was generatedby fusing nineteen (19) α-1,2 mannosidase catalytic domains tothirty-two (32) fungal ER, and cis-Golgi leaders. TABLE 9 Amount ofMan₅GlcNAc₂ on secreted Number K3 protein (% of total glycans) ofconstructs (%) N.D.*  19 (3.1)  0-10% 341 (56.1) 10-20%  50 (8.2) 20-40& 75 (12.3) 40-60%  72 (11.8) More than 60%  51 (8.4)^(†) Total 608 (100)

[0715] Table 7 shows two constructs pFB8 and pGC5, among others, whichenable a transformed host cell to make K3 glycoprotein displayingMan₅GlcNAc₂.

[0716] Table 8 shows a more preferred construct, pBC18-5, a S.cerevisiaeVAN1(s) targeting peptide sequence (from SwissProt 23642) ligatedin-frame to a C. elegans mannosidase IB (Genbank AN CAA98114) with an 80amino acid N-terminal deletion (Saccharomyces Van1(s)/C.elegansmannosidase IB Δ80). This fusion construct also produces a predominantMan₅GlcNAc₂ structure, as shown in FIG. 5E. This mannosidase fusionconstruct was shown to produce greater than 50% Man₅GlcNAc₂ (+++++).

[0717] Generation of a Combinatorial Localization/mannosidase Library:

[0718] Generating a combinatorial DNA library of α-1,2-mannosidasecatalytic domains fused to targeting peptides required the amplificationof mannosidase domains with varying lengths of N-terminal deletions froma number of organisms. To approach this goal, the full length openreading frames (ORFs) of α-1,2-mannosidases were PCR amplified fromeither cDNA or genomic DNA obtained from the following sources: Homosapiens, Mus musculus, Drosophila melanogaster, Caenorhabditis elegans,Aspergillus nidulans and Penicillium citrinum. In each case, DNA wasincubated in the presence of oligonucleotide primers specific for thedesired mannosidase sequence in addition to reagents required to performthe PCR reaction. For example, to amplify the ORF of the M. musculusα-1,2-mannosidase IA, the 5′-primer ATGCCCGTGGGGGGCCTGTTGCCGCTCTTCAGTAGC(SEQ ID NO: 23) and the 3′-primerTCATTTCTCTTTGCCATCAATTTCCTTCTTCTGTTCACGG (SEQ ID NO: 24) were incubatedin the presence of Pfu DNA polymerase (Stratagene, La Jolla, Calif.) andamplified under the conditions recommended by Stratagene using thecycling parameters: 94° C. for 1 min (1 cycle); 94° C. for 30 sec, 68°C. for 30 sec, 72° C. for 3 min (30 cycles). Following amplification theDNA sequence encoding the ORF was incubated at 72° C. for 5 min with 1UTaq DNA polymerase (Promega, Madison, Wis.) prior to ligation intopCR2.1-TOPO (Invitrogen, Carlsbad, Calif.) and transformed into TOP 10chemically competent E. coli, as recommended by Invitrogen. The clonedPCR product was confirmed by ABI sequencing using primers specific forthe mannosidase ORF.

[0719] To generate the desired N-terminal truncations of eachmannosidase, the complete ORF of each mannosidase was used as thetemplate in a subsequent round of PCR reactions wherein the annealingposition of the 5′-primer was specific to the 5′-terminus of the desiredtruncation and the 3′-primer remained specific for the original3′-tenninus of the ORF. To facilitate subcloning of the truncatedmannosidase fragment into the yeast expression vector, pJN347 (FIG. 2C)AscI and PacI restriction sites were engineered onto each truncationproduct, at the 5′- and 3 ′-termini respectively. The number andposition of the N-terminal truncations generated for each mannosidaseORF depended on the position of the transmembrane (TM) region inrelation to the catalytic domain (CD). For instance, if the stem regionlocated between the TM and CD was less than 150 bp, then only onetruncation for that protein was generated. If, however, the stem regionwas longer than 150 bp then either one or two more truncations weregenerated depending on the length of the stem region.

[0720] An example of how truncations for the M. musculus mannosidase 1A(Genbank AN 6678787) were generated is described herein, with a similarapproach being used for the other mannosidases. FIG. 3 illustrates theORF of the M. musculus α-1,2-mannosidase IA with the predictedtransmembrane and catalytic domains being highlighted in bold. Based onthis structure, three 5′-primers were designed (annealing positionsunderlined in FIG. 3) to generate the Δ65-, Δ105- and Δ187-N-tenninaldeletions. Using the Δ65 N-terminal deletion as an example the 5′-primerused was 5′-GGCGCGCCGACTCCTCCAAGCTGCTCAGCGGGGTCCTGTTCCAC-3′ (SEQ ID NO:25) (with the AscI restriction site highlighted in bold) in conjunctionwith the 3′-primer5′-CCTTAATTAATCATTTCTCTTTGCCATCAATTTCCTTCTTCTGTTCACGG-3′ (SEQ ID NO: 26)(with the PacI restriction site highlighted in bold). Both of theseprimers were used to amplify a 1561 bp fragment under the conditionsoutlined above for amplifying the full length M. musculus mannosidase 1AORF. Furthermore, like the product obtained for the full length ORF, thetruncated product was also incubated with Taq DNA polymerase, ligatedinto pCR2.1-TOPO (Invitrogen, Carlsbad, Calif.), transformed into TOP 10and ABI sequenced. After having amplified and confirmed the sequence ofthe truncated mannosidase fragment, the resulting plasmid,pCR2.1-Δ65mMannIA, was digested with AscI and Pacl in New EnglandBiolabs buffer #4 (Beverly, Mass.) for 16 h at 37° C. In parallel, thepJN347 (FIG. 2C) was digested with the same enzymes and incubated asdescribed above. Post-digestion, both the pJN347 (FIG. 2C) back-bone andthe truncated catalytic domain were gel extracted and ligated using theQuick Ligation Kit (New England Biolabs, Beverly, Mass.), as recommendedby the manufacturers, and transformed into chemically competent DH5αcells (Invitrogen, Carlsbad, Calif.). Colony PCR was used to confirm thegeneration of the pJN347-mouse Mannosidase IAΔ65 construct.

[0721] Having generated a library of truncated α-1,2-mannosidasecatalytic domains in the yeast expression vector pJN347 (FIG. 2C) theremaining step in generating the targeting peptide/catalytic domainlibrary was to clone in-frame the targeting peptide sequences (FIG. 2).Both the pJN347-mannosidase constructs (FIG. 2D) and thepCR2.1TOPO-targeting peptide constructs (FIG. 2B) such as were incubatedovernight at 37° C. in New England Biolabs buffer #4 in the presence ofthe restriction enzymes NotI and AscI. Following digestion, both thepJN347-mannosidase back-bone and the targeting peptide regions weregel-extracted and ligated using the Quick Ligation Kit (New EnglandBiolabs, Beverly, Mass.), as recommended by the manufacturers, andtransformed into chemically competent DH5α cells (Invitrogen, Carlsbad,Calif.). Subsequently, the pJN347-targeting peptide/mannosidaseconstructs were ABI sequenced to confirmn that the generated fusionswere in-frame. The estimated size of the final targetingpeptide/alpha-α-1,2-mannosidase library contains over 1300 constructsgenerated by the approach described above. FIG. 2 illustratesconstruction of the combinatorial DNA library.

[0722] Engineering a P.pastoris OCH1 knock-out strain with MultipleAuxotrophic Markers.

[0723] The first step in plasmid construction involved creating a set ofuniversal plasmids containing DNA regions of the KEX1 gene of P.pastoris(Boehm et al. Yeast 1999 May; 15(7):563-72) as space holders for the 5′and 3′ regions of the genes to be knocked out. The plasmids alsocontained the S.cerevisiae Ura-blaster (Alani et al., Genetics 116,541-545. 1987) as a space holder for the auxotrophic markers, and anexpression cassette with a multiple cloning site for insertion of aforeign gene. A 0.9-kb fragment of the P.pastoris KEX1-5′ region wasamplified by PCR using primersGGCGAGCTCGGCCTACCCGGCCAAGGCTGAGATCATTTGTCCAGCTTCA GA (SEQ ID NO: 27) andGCCCACGTCGACGGATCCGTTTAAACATCGATTGGAGAGGCTGACACC GCTACTA (SEQ ID NO: 28)and P.pastoris genomic DNA as a template and cloned into the SacI, SalIsites of pUC19 (New England Biolabs, Beverly, Mass.). The resultingplasmid was cut with BamHI and SalI, and a 0.8-kb fragment of theKEX1-3′ region that had been amplified using primersCGGGATCCACTAGTATTTAAATCATATGTGCGAGTGTACAACTCTTCCC ACATGG (SEQ ID NO: 29)and GGACGCGTCGACGGCCTACCCGGCCGTACGAGGAATTTCTCGG ATGACTCTTTTC (SEQ ID NO:30) was cloned into the open sites-creating pJN262. This plasmid was cutwith BamHI and the 3.8-kb BamHI, BglII fragment of pNKY51 (Alani et al.,Genetics 116, 541-545. 1987) was inserted in both possible orientationsresulting in plasmids pJN263 (FIG. 4A) and pJN284 (FIG. 4B).

[0724] An expression cassette was created with NotI and PacI as cloningsites. The GAPDH promoter of P.pastoris was amplified using primersCGGGATCCCTCGAGAGATCTTTTTTGTAGAAATGTCTTGGTGCCT (SEQ ID NO: 31) andGGACATGCATGCACTAGTGCGGCCGCCACGTGATAGTTGTTCA ATTGATTGAAATAGGGACAA (SEQ IDNO: 32) and plasmid pGAPZ-A (Invitrogen) as template and cloned into theBamHI, SphI sites of pUC19 (New England Biolabs, Beverly, Mass.) (FIG.4B). The resulting plasmid was cut with SpeI and SphI and the CYC1transcriptional terminator region (“TT”) that had been amplified usingprimers CCTTGCTAGCTTAATTAACCGCGGCACGTCCGACGGCGGCCCA CGGGTCCCA (SEQ IDNO: 33) and GGACATGCATGCGGATCCCTTAAGAGCCGGCAGCTTGCAAATTAAAGCCTTCGAGCGTCCC (SEQ ID NO: 34) and plasmid pPICZ-A (Invitrogen) as atemplate was cloned into the open sites creating pJN261 (FIG. 4B).

[0725] A knockout plasmid for the P.pastoris OCH1 gene was created bydigesting pJN263 with SalI and SpeI and a 2.9-kb DNA fragment of theOCH1-5′ region, which had been amplified using the primersGAACCACGTCGACGGCCATTGCGGCCAAAACCTTTTTTCCTATT CAAACACAAGGCATTGC (SEQ IDNO: 35) and CTCCAATACTAGTCGAAGATTATCTTCTACGGTGCCTGGACTC (SEQ ID NO: 36)and P.pastoris genomic DNA as a template, was cloned into the open sites(FIG. 4C). The resulting plasmid was cut with EcoRI and PmeI and a1.0-kb DNA fragment of the OCH1-3′ region that had been generated usingthe primers TGGAAGGTTTAAACAAAGCTAGAGTAAAATAGATATAGCGAG ATTAGAGAATG (SEQID NO: 37) and AAGAATTCGGCTGGAAGGCCTTGTACCTTGATGTAGTTCCCGTT TTCATC (SEQID NO: 38) was inserted to generate pJN298 (FIG. 4C). To allow for thepossibility to simultaneously use the plasmid to introduce a new gene,the BamHI expression cassette of pJN261 (FIG. 4B) was cloned into theunique BamHI site of pJN298 (FIG. 4C) to create pJN299 (FIG. 4E).

[0726] The P.pastoris Ura3-blaster cassette was constructed using asimilar strategy as described in Lu et al. (1998) Appl. Microbiol.Biotechnol. 49:141-146. A 2.0-kb PstI, SpeI fragment of P.pastoris URA3was inserted into the PstI, XbaI sites of pUC19 (New England Biolabs,Beverly, Mass.) to create pJN306 (FIG. 4D). Then a 0.7-kb SacI, PvuIIDNA fragment of the lacZ open reading frame was cloned into the SacI,SmaI sites to yield pJN308 (FIG. 4D). Following digestion of pJN308(FIG. 4D) with Pstl, and treatment with T4 DNA polymerase, theSacI-PvuII fragment from lacZ that had been blunt-ended with T4 DNApolymerase was inserted generating pJN315 (FIG. 4D). The lacZ/URA3cassette was released by digestion with SacI and SphI, blunt ended withT4 DNA polymerase and cloned into the backbone of pJN299 that had beendigested with PmeI and AflII and blunt ended with T4 DNA polymerase. Theresulting plasmid was named pJN329 (FIG. 4E).

[0727] A HIS4 marked expression plasmid was created by cutting pJN261(FIG. 4F) with EcoICRI (FIG. 4F). A 2.7 kb fragment of the Pichiapastoris HIS4 gene that had been amplified using the primersGCCCAAGCCGGCCTTAAGGGATCTCCTGATGACTGACTCACTGATAATA AAAATACGG (SEQ ID NO:39) and GGGCGCGTATTTAAATACTAGTGGATCTATCGAATCTAAATGTAAGTTA AAATCTCTAA(SEQ ID NO: 40) cut with NgoMIV and Swal and then blunt-ended using T4DNA polymerase, was then ligated into the open site. This plasmid wasnamed pJN337 (FIG. 4F). To construct a plasmid with a multiple cloningsite suitable for fusion library construction, pJN337 was cut with NotIand PacI and the two oligonucleotidesGGCCGCCTGCAGATTTAAATGAATTCGGCGCGCCTTAAT (SEQ ID NO: 41) andTAAGGCGCGCCGAATTCATTTAAATCTGCAGGGC (SEQ ID NO: 42), that had beenannealed in vitro were ligated into the open sites, creating pJN347(FIG. 4F).

[0728] To create an och1 knockout strain containing multiple auxotrophicmarkers, 100 μg of pJN329 was digested with SfiI and used to transformP.pastoris strain JC308 (Cereghino et al. Gene 263 (2001) 159-169) byelectroporation.

[0729] Following transformiation, the URA dropout plates were incubatedat room temperature for 10 days. One thousand (1000) colonies werepicked and restreaked. All 1000 clones were then streaked onto 2 sets ofURA dropout plates. One set was incubated at room temperature, whereasthe second set was incubated at 37° C. The clones that were unable togrow at 37° C., but grew at room temperature, were subjected to colonyPCR to test for the correct OCH1 knockout. One clone that showed theexpected PCR signal (about 4.5 kb) was designated YJN153.

EXAMPLE 5 Characterization of the Combinatorial Localization/MannosidaseLibrary

[0730] Positive transformants (Example 4) screened by colony PCR toconfirm integration of the mannosidase construct into the P.pastorisgenome were subsequently grown at room temperature in 50 ml BMGYbuffered methanol-complex medium consisting of 1% yeast extract, 2%peptone, 100 mM potassium phosphate buffer, pH 6.0, 1.34% yeast nitrogenbase, 4×10⁻⁵% biotin, and 1% glycerol as a growth medium) untilOD_(600nm) 2-6 at which point they were washed with 10 ml BMMY (bufferedmethanol-complex medium consisting of 1% yeast extract, 2% peptone, 100mM potassium phosphate buffer, pH 6.0, 1.34% yeast nitrogen base,4×10⁻⁵% biotin, and 1.5% methanol as a growth medium) media prior toinduction of the reporter protein for 24 hours at room temperature in 5ml BMMY. Consequently, the reporter protein was isolated and analyzed asdescribed in Example 3 to characterize its glycan structure. Using thetargeting peptides in Table 6, mannosidase catalytic domains localizedto either the ER or the Golgi showed significant level of trimming of aglycan predominantly containing Man₈GlcNAc₂ to a glycan predominantlycontaining Man₅GlcNAc₂. This is evident when the glycan structure of thereporter glycoprotein is compared between that of P.pastoris och1knock-out in FIGS. 5C and 6C and the same strain transformed with M.musculus mannosidase constructs as shown in FIGS. 5D, 5E, 6D-6F. FIGS. 5and 6 show expression of constructs generated from the combinatorial DNAlibrary which show significant mannosidase activity in P.pastoris.Expression of pGC5 (Saccharomyces MNS1(m)/mouse mannosidase IB Δ99)(FIGS. 5D and 6E) produced a protein which has approximately 30% of allglycans trimmed to Man₅GlcNAc₂, while expression of pFB8 (SaccharomycesSEC12(m)/mouse mannosidase IA Δ187) (FIG. 6F) produced approximately 50%Man₅GlcNAc₂ and expression of pBC18-5 (Saccharomyces VAN1(s)/C. elegansmannosidase IB Δ80) (FIG. 5E) produced 70% Man₅GlcNAc₂.

EXAMPLE 6 Trimming in vivo by Alpha-1,2-mannosidase

[0731] To ensure that the novel engineered strains of Example 4 in factproduced the desired Man₅GlcNAc₂ structure in vivo, cell supernatantswere tested for mannosidase activity (see FIGS. 7-9). For eachconstruct/host strain described below, HPLC was performed at 30° C. witha 4.0mm×250 mm column of Altech (Avondale, Pa., USA) Econosil-NH₂ resin(5 μm) at a flow rate of 1.0 ml/min for 40 min. In FIGS. 7 and 8,degradation of the standard Man₉GlcNAc₂ [b] was shown to occur resultingin a peak which correlates to Man₈GlcNAc₂. In FIG. 7, the Man₉GlcNAc₂[b] standard eluted at 24.61 min and Man₅GlcNAc₂ [a] eluted at 18.59min. In FIG. 8, Man₉GlcNAc₂ eluted at 21.37 min and Man₅GlcNAc₂ at 15.67min. In FIG. 9, the standard Man₈GlcNAc₂ [b] was shown to elute at 20.88min.

[0732]P.pastoris cells comprising plasmid pFB8 (Saccharomyces SEC12(m)/mouse mannosidase IA Δ187) were grown at 30° C. in BMGY to an OD600of about 10. Cells were harvested by centrifugation and transferred toBMMY to induce the production of K3 (kringle 3 from human plasminogen)under control of an AOXI promoter. After 24 hours of induction, cellswere removed by centrifugation to yield an essentially clearsupernatant. An aliquot of the supernatant was removed for mannosidaseassays and the remainder was used for the recovery of secreted solubleK3. A single purification step using CM-sepharose chromatography and anelution gradient of 25 mM NaAc, pH5.0 to 25 mM NaAc, pH5.0, 1M NaCl,resulted in a 95% pure K3 eluting between 300-500 mM NaCl. N-glycananalysis of the K3 derived glycans is shown in FIG. 6F. The earlierremoved aliquot of the supernatant was further tested for the presenceof secreted mannosidase activity. A commercially available standard of2-aminobenzamide-labeled N-linked-type oligomannose 9 (Man9-2-AB)(Glyko, Novato, Calif.) was added to: BMMY (FIG. 7A), the supernatantfrom the above aliquot (FIG. 7B), and BMMY containing 10 ng of 75 mU/mLof α-1,2-mannosidase from Trichoderma reesei (obtained from Contreras etal., WO 02/00856 A2) (FIG. 7C). After incubation for 24 hours at roomtemperature, samples were analyzed by amino silica HPLC to determine theextent of mannosidase trimming.

[0733]P.pastoris cells comprising plasmid pGC5 (SaccharomycesMNS1(m)/mouse mannosidase IB Δ99) were similarly grown and assayed.Cells were grown at room temperature in BMGY to an OD600 of about 10.Cells were harvested by centrifugation and transferred to BMMY to inducethe production of K3 under control of an AOXI promoter. After 24 hoursof induction, cells were removed by centrifugation to yield anessentially clear supernatant. An aliquot of the supernatant was removedfor mannosidase assays and the remainder was used for the recovery ofsecreted soluble K3. A single purification step using CM-sepharosechromatography and an elution gradient of 25 mM NaAc, pH5.0 to 25 mMNaAc, pH5.0, 1M NaCl, resulted in a 95% pure K3 eluting between 300-500mM NaCl. N-glycan analysis of the K3 derived glycans is shown in FIG.5D. The earlier removed aliquot of the supernatant was further testedfor the presence of secreted mannosidase activity as shown in FIG. 8B. Acommercially available standard of Man9-2-AB (Glyko, Novato, Calif.)were added to: BMMY (FIG. 8A), supernatant from the above aliquot (FIG.8B), and BMMY containing 10 ng of 75 mU/mL of α-1,2-mannosidase fromTrichoderma reesei (obtained from Contreras et al., WO 02/00856 A2)(FIG. 8C). After incubation for 24 hours at room temperature, sampleswere analyzed by amino silica HPLC to determine the extent ofmannosidase trimming.

[0734] Man9-2-AB was used as a substrate and it is evident that after 24hours of incubation, mannosidase activity was virtually absent in thesupernatant of the pFB8 (Saccharomyces SEC12 (m)/mouse mannosidase IAΔ187) strain digest (FIG. 7B) and pGC5 (Saccharomyces MNS1(m)/mousemannosidase IB Δ99) strain digest (FIG. 8B) whereas the positive control(purified α-1,2-mannosidase from T. reesei obtained from Contreras)leads to complete conversion of Man₉GlcNAc₂ to Man₅GlcNAc₂ under thesame conditions, as shown in FIGS. 7C and 8C. This is conclusive datashowing in vivo mannosidase trimming in P.pastoris pGC5 strain; and pFB8strain, which is distinctly different from what has been reported todate (Contreras et al., WO 02/00856 A2).

[0735]FIG. 9 further substantiates localization and activity of themannosidase enzyme. P.pastoris comprising pBC18-5 (SaccharomycesVAN1(s)/C.elegans mannosidase IB Δ80) was grown at room temperature inBMGY to an OD600 of about 10. Cells were harvested by centrifugation andtransferred to BMMY to induce the production of K3 under control of anAOXI promoter. After 24 hours of induction, cells were removed bycentrifugation to yield an essentially clear supernatant. An aliquot ofthe supernatant was removed for mannosidase assays and the remainder wasused for the recovery of secreted soluble K3. A single purification stepusing CM-sepharose chromatography and an elution gradient 25 mM NaAc,pH5.0 to 25 mM NaAc, pH5.0, 1M NaCl, resulted in a 95% pure K3 elutingbetween 300-500 mM NaCl. N-glycan analysis of the K3 derived glycans isshown in FIG. 5E. The earlier removed aliquot of the supernatant wasfurther tested for the presence of secreted mannosidase activity asshown in FIG. 9B. A commercially available standard of Man8-2-AB (Glyko,Novato, Calif.) was added to: BMMY (FIG. 9A), supernatant from the abovealiquot pBC18-5 (Saccharomyces VAN1(s)/ C. elegans mannosidase IB Δ80)(FIG. 9B), and BMMY containing media from a different fusion constructpDD28-3 (Saccharomyces MNN10(m) (from SwissProt 50108)/H. sapiensmannosidase IB Δ99) (FIG. 9C). After incubation for 24 hours at roomtemperature, samples were analyzed by amino silica HPLC to determine theextent of mannosidase trimming. FIG. 9B demonstrates intracellularmannosidase activity in comparison to a fusion construct pDD29-3(Saccharomyces MNN10(m) H. sapiens mannosidase IB Δ99) exhibiting anegative result (FIG. 9C).

EXAMPLE 7 pH Optimum Assay of an Engineered α-1,2-mannosidase

[0736]P.pastoris cells comprising plasmid pBB27-2 (Saccharomyces MNN10(s) (from SwissProt 50108)/C. elegans mannosidase IB Δ31) were grown atroom temperature in BMGY to an OD600 of about 17. About 80 μL of thesecells were inoculated into 600 μL BMGY and were grown overnight.Subsequently, cells were harvested by centrifugation and transferred toBMMY to induce the production of K3 (kringle 3 from human plasminogen)under control of an AOX1 promoter. After 24 hours of induction, cellswere removed by centrifugation to yield an essentially clear supernatant(pH 6.43). The supernatant was removed for mannosidase pH optimumassays. Fluorescence-labeled Man₈GlcNAc₂ (0.5 μg) was added to 20 μL ofsupernatant adjusted to various pH (FIG. 11) and incubated for 8 hoursat room temperature. Following incubation the sample was analyzed byHPLC using an Econosil NH2 4.6×250 mm, 5 micron bead, amino-bound silicacolumn (Altech, Avondale, Pa.). The flow rate was 1.0 ml/min for 40 minand the column was maintained to 30° C. After eluting isocratically (68%A:32% B) for 3 min, a linear solvent gradient (68% A:32% B to 40% A:60%B) was employed over 27 min to elute the glycans (18). Solvent A(acetonitrile) and solvent B (ammonium formate, 50 mM, pH 4.5. Thecolumn was equilibrated with solvent (68% A:32% B) for 20 min betweenruns.

EXAMPLE 8 Engineering of P.pastoris to Produce N-glycans with theStructure GlcNAcMan₅GlcNAc₂

[0737] GlcNAc Transferase I activity is required for the maturation ofcomplex and hybrid N-glycans (U.S. Pat. No. 5,834,251). Man₅GlcNAc₂ mayonly be trimmed by mannosidase II, a necessary step in the formation ofhuman glycoforms, after the addition of N-acetylglucosamine to theterminal α-1,3 mannose residue of the trimannose stem by GlcNAcTransferase I (Schachter, 1991 Glycobiology 1(5):453-461). Accordingly,a combinatorial DNA library was prepared including DNA fragmentsencoding suitably targeted catalytic domains of GlcNAc Transferase Igenes from C. elegans and Homo sapiens; and localization sequences fromGLS, MNS, SEC, MNN9, VAN1, ANP1, HOC1, MNN10, MNN11, MNT1, KTR1, KTR2,MNN2, MNN5, YUR1, MNN1, and MNN6 from S.cerevisiae and P.pastorisputative α-1,2-mannosyltransferases based on the homology fromS.cerevisiae: D2, D9 and J3, which are KTR homologs. Table 10 includesbut does not limit targeting peptide sequences such as SEC and OCH1,from P.pastoris and K.lactis GnTI, (See Table 6 and Table 10). TABLE 10A representative combinatorial library of targeting peptidesequences/catalytic domain for UDP-N-Acetylglucosaminyl Transferase I(GnTI) Catalytic Targeting peptide Domain OCH1(s) OCH1(m) OCH1(l)MNN9(s) MNN9(m) Human, PB105 PB106 PB107 PB104 N/A GnTI, Δ38 Human, NB12NB13 NB14 NB15 NB GnTI, Δ86 C. elegans, OA12 OA13 OA14 OA15 OA16 GnTI,Δ88 C. elegans, PA12 PA13 PA14 PA15 PA16 GnTI, Δ35 C. elegans, PB12 PB13PB14 PB15 PB16 GnTI, Δ63 X. leavis, QA12 QA13 QA14 QA15 QA16 GnTI, Δ33X. leavis, QB12 QB13 QB14 QB15 QB16 GnTI, Δ103

[0738] Targeting peptide sequences were selected from OCH1 in P.pastoris(long, medium and short) (see Example 4) and MNN9 (SwissProt P39107) inS.cerevisiae short, and medium. Catalytic domains were selected fromhuman GnTI with a 38 and 86 amino acid N-terminal deletion, C. elegans(gly-12) GnTI with a 35 and 63 amino acid deletion as well as C. elegans(gly-14) GnTI with a 88 amino acid N-terminal deletion and X. leavisGnTI with a 33 and 103 amino acid N-terminal deletion, respectively.

[0739] A portion of the gene encoding human N-acetylglucosaminylTransferase] (MGATI, Accession# NM002406), lacking the first 154 bp, wasamplified by PCR using oligonucleotides5′-TGGCAGGCGCGCCTCAGTCAGCGCTCTCG-3′ (SEQ ID NO: 43) and 5′-AGGTTAATTAAGTGCTAATTCCAGCTAGG-3′ (SEQ ID NO: 44) and vector pHG4.5 (ATCC# 79003)as template. The resulting PCR product was cloned into pCR2.1-TOPO andthe correct sequence was confirmed. Following digestion with AscI andPacI the truncated GnTI was inserted into plasmid pJN346 to create pNA.After digestion of pJN271 with NotI and AscI, the 120 bp insert wasligated into pNA to generate an in-frame fusion of the MNN9transmembrane domain with the GnTI, creating pNA 15.

[0740] The host organism is a strain of P.pastoris that is deficient inhypernannosylation (e.g. an och1 mutant), provides the substrateUDP-GlcNAc in the Golgi and/or ER (i.e. contains a functional UDP-GlcNActransporter), and provides N-glycans of the structure Man₅GlcNAc₂ in theGolgi and/or ER (e.g. P.pastoris pFB8 (Saccharomnyces SEC12 (m)/mousemannosidase IA Δ187) from above). First, P.pastoris pFB8 was transformedwith pPB103 containing the Kluyveromyces lactis MNN2-2 gene (Genbank ANAF106080) (encoding UDP-GlcNAc transporter) cloned into BamHI and BglIIsite of pBLADE-SX plasmid (Cereghino et al. Gene 263 (2001) 159-169).Then the aforementioned combinatorial DNA library encoding a combinationof exogenous or endogenous GnTI/localization genes was transformed andcolonies were selected and analyzed for the presence of the GnTIconstruct by colony PCR. Our transformation and integration efficiencywas generally above 80% and PCR screening can be omitted once robusttransformation parameters have been established.

[0741] In summary, the methods of the invention yield strains ofP.pastoris that produce GlcNAcMan₅GlcNAc₂ in high yield, as shown inFIG. 10B. At least 60% of the N-glycans are GlcNAcMan₅GlcNAc₂. To date,no report exists that describes the formation of GlcNAcMan₅GlcNAc₂ onsecreted soluble glycoproteins in any yeast. Results presented hereinshow that addition of the UDP-GlcNAc transporter along with GnTIactivity produces a predominant GlcNAcMan₅GlcNAc₂ structure, which isconfirmed by the peak at 1457 (m/z) (FIG. 10B).

[0742] Construction of Strain PBP-3:

[0743] The P.pastoris strain expressing K3, (Δoch1, arg-, ade-, his-)was transformed successively with the following vectors. First, pFB8(Saccharomyces SEC12 (m)/mouse mannosidase IA Δ187) was transformed inthe P.pastoris strain by electroporation. Second, pPB103 containingKluyveromyces lactis MNN2-2 gene (Genbank AN AF106080) (encodingUDP-GlcNAc transporter) cloned into pBLADE-SX plasmid (Cereghino et al.Gene 263 (2001) 159-169) digested with BamHI and BglII enzymes wastransformed in the P.pastoris strain. Third, pPB104 containingSaccharomyces MNN9(s)/human GnTI Δ38 encoding gene cloned as NotI-PacIfragment into pJN336 was transformed into the P.pastoris strain.

EXAMPLE 9 Engineering K.lactis Cells to Produce N-glycans with theStructure MansGlcNAc₂

[0744] Identification and Disruption of the K.lactis OCH1 gene

[0745] The OCH1 gene of the budding yeast S.cerevisiae encodes a1,6-mannosyltransferase that is responsible for the first Golgilocalized mannose addition to the Man₈GlcNAc₂N-glycan structure onsecreted proteins (Nakanishi-Shindo et al. (1993), J. Biol. Chem.;268(35):26338-45). This mannose transfer is generally recognized as thekey initial step in the fungal specific polymannosylation of N-glycanstructures (Nakanishi-Shindo et al. (1993) J. Biol. Chem.268(35):26338-26345; Nakayama et al. (1992) EMBO J. 11(7):2511-19;Morin-Ganet et al, Traffic 1(1):56-68. (January 2000)). Deletion of thisgene in S.cerevisiae results in a significantly shorter N-glycanstructure that does not include this typical polymannosylation or agrowth defect at elevated temperatures (Nakayama et al. (1992) EMBO J.11(7):2511-19).

[0746] The Och1p sequence from S.cerevisiae was aligned with knownhomologs from Candida albicans (Genbank accession # AAL49987), andP.pastoris along with the HocI proteins of S.cerevisiae (Neiman et al,Genetics, 145(3):637-45 (March 1997) and K.lactis (PENDANT EST database)which are related but distinct mannosyltransferases. Regions of highhomology that were in common among Och1p homologs but distinct from theHoc1p homologs were used to design pairs of degenerate primers that weredirected against genomic DNA from the K.lactis strain MG1/2 (Bianchi etal, Current Genetics 12, 185-192 (1987)). PCR amplification with primersRCD33 (CCAGAAGAATTCAATTYTGYCARTGG) (SEQ ID NO: 45) and RCD34(CAGTGAAAATACCTGGNCCNGTCCA) (SEQ ID NO: 46) resulted in a 302 bp productthat was cloned and sequenced and the predicted translation was shown tohave a high degree of homology to Och1 proteins (>55% to S.cerevisiaeOch1p).

[0747] The 302 hp PCR product was used to probe a Southern blot ofgenomic DNA from K.lactis strain (MG1/2) with high stringency (Sambrooket al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1989). Hybridizationwas observed in a pattern consistent with a single gene indicating thatthis 302 bp segment corresponds to a portion of the K.lactis genome andK.lactis (KlOCH1) contains a single copy of the gene. To clone theentire KlOCH1 gene, the Southern blot was used to map the genomic locus.Accordingly, a 5.2 kb BamHI/PstI fragment was cloned by digestinggenomic DNA and ligating those fragments in the range of 5.2 kb intopUC19 (New England Biolabs, Beverly, Mass.) to create a K.lactissubgenomic library. This subgenomic library was transformed into E. coliand several hundred clones were tested by colony PCR using RCD 33/34.The 5.2 kb clone containing the predicted KlOCH1 gene was sequenced andan open reading frame of 1362 bp encoding a predicted protein that is46.5% identical to the S.cerevisiae OCH1 gene. The 5.2 kb sequence wasused to make primers for construction of an och1:KAN^(R) deletion alleleusing a PCR overlap method (Davidson et al. (2002) Microbiol. 148(Pt8):2607-15). This deletion allele was transformed into two K.lactisstrains and G418 resistant colonies selected. These colonies werescreened by both PCR and for temperature sensitivity to obtain a straindeleted for the OCH1 ORF. The results of the experiment show strainswhich reveal a mutant PCR pattern, which were characterized by analysisof growth at various temperatures and N-glycan carbohydrate analysis ofsecreted and cell wall proteins following PNGase digestion. The och1mutation conferred a temperature sensitivity which allowed strains togrow at 30° C. but not at 35° C. FIG. 12A shows a MALDI-TOF analysis ofa wild type K.lactis strain producing N-glycans of Man₈GlcNAc₂ [c] andhigher.

[0748] Identification, Cloning, and Disruption of the K.lactis MNN1 gene

[0749]S.cerevisiae MNN1 is the structural gene for the Golgiα-1,3-mannosyltransferase. The product of MNN1 is a 762-amino acid typeII membrane protein (Yip et al., Proc Natl Acad Sci U S A. 91(7):2723-7.(1994)). Both N-linked and O-linked oligosaccharides isolated from mnn1mutants lack α-1,3-mannose linkages (Raschke et al., J Biol Chem.,248(13):4660-6. (Jul. 10, 1973).

[0750] The Mnn1p sequence from S.cerevisiae was used to search theK.lactis translated genomic sequences (PEDANT). One 405 bp DNA sequenceencoding a putative protein fragment of significant similarity to Mnn1pwas identified. An internal segment of this sequence was subsequentlyPCR amplified with primers KMN1 (TGCCATCTTTTAGGTCCAGGCCCGTTC) (SEQ IDNO: 47) and KMN2 (GATCCCACGACGCATCGTATTTCTTTC), (SEQ ID NO: 48) and usedto probe a Southern blot of genomic DNA from K.lactis strain (MG1/2).Based on the Southern hybridization data a 4.2 Kb BamHI-PstI fragmentwas cloned by generating a size-selected library as described herein. Asingle clone containing the K.lactis MNN1 gene was identified by wholecolony PCR using primers KMN1 (SEQ ID NO: 47) and KMN2 (SEQ ID NO: 48)and sequenced. Within this clone a 2241 bp ORF was identified encoding apredicted protein that was 34% identical to the S.cerevisiae MNN1 gene.Primers were designed for construction of a mnn1::NAT^(R) deletionallele using the PCR overlap method (Davidson et al. (2002) Microbiol.148(Pt 8):2607-15).

[0751] This disruption allele was transformed into a strain of K.lactisby electroporation and nourseothricin resistant transformants wereselected and PCR amplified for homologous insertion of the disruptionallele. Strains that reveal a mutant PCR pattern may be subjected toN-glycan carbohydrate analysis of a known reporter gene.

[0752]FIG. 12B depicts the N-glycans from the K.lactis och1 mnn1deletion strain observed following PNGase digestion the MALDI-TOF asdescribed herein. The predominant peak at 1908 (m/z) indicated as [d] isconsistent with the mass of Man₉GlcNAc₂.

[0753] Additional methods and reagents which can be used in the methodsfor modifying the glycosylation are described in the literature, such asU.S. Pat. No. 5,955,422, U.S. Pat. No. 4,775,622, U.S. Pat. No.6,017,743, U.S. Pat. No. 4,925,796, U.S. Pat. No. 5,766,910, U.S. Pat.No. 5,834,251, U.S. Pat. No. 5,910,570, U.S. Pat. No. 5,849,904, U.S.Pat. No. 5,955,347, U.S. Pat. No. 5,962,294, U.S. Pat. No. 5,135,854,U.S. Pat. No. 4,935,349, U.S. Pat. No. 5,707,828, and U.S. Pat. No.5,047,335. Appropriate yeast expression systems can be obtained fromsources such as the American Type Culture Collection, Rockville, Md.Vectors are commercially available from a variety of sources.

EXAMPLE 10 Strains, Culture Conditions and Reagents

[0754] For the examples below, the following strains, culture conditionsand reagents were used. Escherichia coli strains TOP10 or DH5α were usedfor recombinant DNA work.

[0755] Protein expression was carried out at room temperature in a96-well plate fornat with buffered glycerol-complex medium (BMGY)consisting 1% yeast extract, 2% peptone, 100 mM potassium phosphatebuffer, pH 6.0, 1.34% yeast nitrogen base, 4×10⁻⁵% biotin, and 1%glycerol as a growth medium. The induction medium was bufferedmethanol-complex medium (BMMY) consisting of 1.5% methanol instead ofglycerol in BMGY.

[0756] Restriction and modification enzymes were from New EnglandBioLabs (Beverly, Mass.).

[0757] Oligonucleotides were obtained from the Dartmouth College Corefacility (Hanover, N.H.) or Integrated DNA Technologies (Coralville,Iowa).

EXAMPLE 11 Cloning And Generation Of Expression Vectors To ProduceMan₃GlcNAc₂

[0758] Restriction and modification enzymes were from New EnglandBioLabs (Beverly, Mass.). The shuttle vector pVM2 was generated frompUC19 by inverse PCR (Sambrook, J., Fritsch, E. F., and Maniatis, T.(1989)ln: Molecular Cloning, a Laboratory Manual 2nd Edition, ColdSpring Harbor N.Y.: Cold Spring Harbor Laboratory Press.) using theprimers VJM104 and VJM106 (5′-GCGGCCGCGGATCCCCGGGTACCGAGCTCGAATTCACT-3′and 5′-GGGGCGCGCC TTAATTAACGACCTGCAGGCATGCAAGCTTGGCGTAATCATGGTCAT-3′respectively, introduced restriction sites NotI, AscI and PacI areunderlined).

[0759] The roll-in plasmid pJN285 is a derivative of the knock-inplasmid pJN266 that was constructed in the following way. A 0.9-kbfragment of the PpKEXI-5′ region was amplified by PCR using primersKex55 (5′-GGCGAGCTCGGCCTACCCGGCCAAGGCTGAGATCATTTGTCCAG CTTCAGA -3′) andKex53 (5′-GCCCACGTCGACGGATCCGTTTAAACATCGATTGGAG AGGCTGACACCGCTACTA-3═)from Pichia pastoris genomic DNA and cloned into pUC19 digested withSacI and SalI. The resulting plasmid was cut with BamHI and SalI, and a0.8-kb fragment of the KEX1-3′ region that had been amplified usingprimers Kex35 (5′-CGGGATCCACTAGTATTTAAATCATATGTGCGAGTGTACAACTCTTCCCACATGG-3′) and Kex33 (5′-GGACGCGTCGACGGCCTACCCGGCCGTACGAGGAATTTCTCGGATGACTCTTTTC -3′) was cloned into pJN262 digested with the same enzymes.This plasmid was cut with BamHI and the 3.8-kb BamHI-BglII fragment ofpNKY51 (1)was inserted in each of the two possible orientationsresulting in plasmids pJN263 and pJN264. To create an expressioncassette with NotI and PacI cloning sites, the GAPDH promoter of P.pastoris was amplified using primers Gap5(5′-CGGGATCCCTCGAGAGATCTTTTTTGTAGAAATGTCTTGGTGCCT-3′) and Gap3(5′-GGACATGCATGCACTAGTGCGGCCGCCACGTGATAGTTGTTCA ATTGATTGAAATAGGGACAA-3′)and plasmid pGAPZ-A (Invitrogen) as template and cloned into theBamHI-SphI sites of pUC19. The resulting plasmid was cut with SpeI andSphI and the S. cerevisiae CYC1 transcriptional terminator region, thathad been amplified from pPICZ-A (Invitrogen) using primers Cyc5(5′-CCTTGCTAGCTTAATTAACC GCGGCACGTCCGACGGCGGCCCACGGGTCCCA-3′) and Cyc3(5′-GGACATGCATGCGGATCCCTTAAGAGCCGGCAGCTTGCAAATTAAAGCCTTCGAGCGTC CC-3′),was cloned into the open sites creating pJN261. The GAPDH/CYC1expression cassette was released by BamHI digestion and cloned eitherinto pJN263 resulting in plasmid pJN265, or into pJN264 resulting inplasmids pJN266 and pJN267 (depending on orientation of the insert).Subsequently the plasmid pJN266 was cut with NgoMIV and Swal to releasethe URA-blaster cassette, and a NgoMIV-SwaI fragment containing thePpHIS4 gene, that had been amplified from pPIC3.5 (Invitrogen) usingprimers JNHIS1 (5′-GCCCAAGCCGGCCTTAAGGGATCTCCTGATGACTGACTCACTGATAATAAAAATACGG-3′) and JNHIS2(5′-GGGCGCGTATTTAAATACTAGTGGATCTATCGAATCTAAATGTAAGTTAAAATCTCTAA-3′), wascloned into the open sites to create pJN285.

[0760] The pJN348 expression vector is based on plasmid pBLURA-SX (2).First a BamHI fragment containing the GAPDH/CYC1 expression cassettefrom vector pJN261 was cloned into pBLURA-SX that had been cut withBamHI and BglII to create plasmid pJN338. Subsequently the latterplasmid was cut with NotI and PacI and the two, oligonucleotides Expr1(5′-GGCCGCCTGCAGATTTAAATGAATTCGGCGCGCCTTAAT-3′) and Expr2(5′-TAAGGCGCGCCGAATTCATTTAAATCTGCAGGGC-3′, the restriction site AscI is,underlined) that had been annealed in vitro, were ligated into the opensites, to create pJN348. 106931 The pPB124 expression vector wasconstructed in several steps based on pBLADE-SX vector described byCereghino et al. Gene 263 (2001) 159-169. First, BamHI fragmentcontaining GAPDH/CYC1 expression cassette from vector pJN261 (describedin Choi et al. Proc Natl Acad Sci U S A. 2003 Apr. 29; 100(9):5022-7)was cloned into pBLADE-SX vector after BamHI-BglII digest. Next, theXhol-NotI fragment containing P. pastoris GAPDH promoter was replacedwith the promoter of P. pastoris PMA1 gene that was amplified with PMA1(5′-TTCCTCGAGATTCAAGCGAATGAGAATAATG-3′) and PMA2 (5′-TTGCGGCCGCGAAGTTTTTAAAGGAAAGAGATA-3′) primers. The resulting vector was then digestedwith XbaI-BamHI enzymes to remove ADE1 marker, and after fill-inreaction ligated with blunt-ended BglII-SacI fragment containingnourseothricin resistance marker from vector pAG25 (Goldstein andMcCusker, Yeast. 1999 October;15(14):1541-53).

EXAMPLE 12 Generation of Localization Signal/Mannosidase I CatalyticDomain Fusions

[0761] Amplificaiion of mouse mannosidase 1A. The gene sequence encodingthe catalytic domain of mouse mannosidase 1A (Genbank: NM_(—)008548, Lal& Moremen 1994) was amplified from mouse liver cDNA (Clontech). Briefly,the forward primer mMIAΔ187-AscI and reverse primer mMIA-PacI(5′-GGCGCGCCGAGCCCGCTGACGCCACCATCCGTGAGAAGAGG GC-3′ and5′-CCTTAATTAATCATTTCTCTTTGCCATCAATTTCCTTCTTCTGTTCACGG-3′, respectively,introduced AscI and PacI restriction sites are underlined) where used toamplify amino acids 188-655 of the mouse mannosidase IA ORF from mouseliver cDNA (Clontech) with Pfu DNA polymerase (Stratagene). Theconditions used for thermo cycling were: 94° C. for 1 min, 1 cycle; 94°C. for 30 sec, 68° C. for 30 sec, 72° C. for 3 min, 30 cycles.Subsequently, 1 μl Taq DNA polymerase (Promega) was added and thereaction further incubated at 72° C. for 10 min with the 1.4 Kb productbeing ligated into pCR2. 1, giving the plasmid pSH9. Followingconfinnation of the PCR product by Taq DyeDeoxy terminal sequencing themouse mannosidase IA was digested with the restriction enzymes AscI andPacI prior to subcloning into the vector pVM2, digested with the samerestriction enzymes, generating the plasmid pSH21.

[0762] To facilitate the subsequent localization of the truncated mousemannosidase IA to the yeast Golgi a region of the S.cerevisiae Sec12protein (amino acids 331-432, encoding the transmembrane domain) wasamplified with the primers SC125 and SC122(5′-ATGTGGCGGCGGCCGCCACCATGAACACTATCCACATAATAAAATTAC CGCTTAACTACGCC-3′and 5′-GGCGCGCCCCACGCCTAGCACTTTTATGGAATCTACGCTAGGTAC-3′, respectively,introduced NotI and AscI restricition sites are underlined) in thepresence of Taq DNA polymerase and S.cerevisiae genomic DNA, producingthe plasmid pJN305. Following confirmation of the PCR product by TaqDyeDeoxy terminal sequencing the Sec12 fragment, digested with therestriction enzymes NotI and AscI, was subcloned into pSH21 digestedwith the same enzymes, generating the plasmid pSH29. Subsequently theNotI/PacI fragment of pSH29, encoding the Sec12 fragment in-frame withthe truncated mouse mannosidase IA, was subcloned into pJN285 digestedwith the same enzymes, generating the plasmid pFB8.

EXAMPLE 13

[0763] Generation of Mannosidase II construct

[0764] The catalytic domain of a Drosophila mannosidase II (GenBank:X77652, Foster and Roberts 1995), encoding amino acids 75-1108, wasamplified from Drosophila ovary cDNA using ExTaq DNA polymerase underthe thermocycling conditions outlined above, by annealing at 55° C. andextending for 5 minutes. The forward primer dMannIIΔ74_AscI and thereverse primer dMannII_PacI (5′-GGCGCGCCCGCGACGATCCAATAAGACCTCCAC-3′ and5′-CCTTAATTAATCAGCTTG AGTGACTGCTCACATAAGCGGCGG-3′, respectively,introduced AscI and PacI restriction sites are underlined) were used.Following confirmation of the PCR product by Taq DyeDeoxy terminalsequencing, the plasmid was named pSH214. Subsequently, the Drosophilamannosidase II fragment was removed from this plasmid by digestion withthe restriction enzymes AscI and PacI, and subcdoned into pJN348digested with the same enzymes, generating the plasmid pSH220.

[0765] To facilitate the subsequent localization of the truncatedDrosophila mannosidase II domain to the Golgi, a region of theS.cerevisiae Mnn2 protein (amino acids 1-36, encoding the transmembranedomain) was amplified with the primers Mnn25 and Mnn21(5′-AGTAAAATGCGGCCGCCACCATGCTGCTTACCAAAAGGTTTTCAAAGC TGTTC-3′ and5′-GGCGCGCCCCGACGTGTTCTCATCCATGTATTTGTTTGTAATGAC-3′, respectively,introduced NotI and AscI restricition sites are underlined) in thepresence of Taq DNA polymerase and S.cerevisiae genomic DNA, producingthe plasmid pJN281. Following confirmnation of the PCR product by TaqDyeDeoxy terminal sequencing, the Mnn2 fragment was digested with therestriction enzymes NotI and AscI and subcloned into pSH220 digestedwith the same enzymes, producing an in-frame fusion of the Mnn2localization signal with the Drosophila mannosidase II catalytic domain,generating the plasmid pKD53. The pH optimum of this engineeredDrosophila mannosidase II catalytic domain was determined to be pH 6.2using a pH assay essentially as described in Example 7.

EXAMPLE 14 Mannosidase II Catalytic Domain Library

[0766] The library of mannosidase II catalytic domains and leadersshowing activity are shown below in Table II. The number of (+)s, asused herein, indicates the relative levels of GlcNAcMan₃GlcNA₂production of % neutral glycans. The notation (−) indicates no apparentproduction of GlcNAcMan₃GlcNA₂. The notation (+) indicates less than 20%production of GlcNAcMan₃GlcNA₂. The notation (++) indicates about 20-30%production of GlcNAcMan₃GlcNA₂. The notation with (+++) indicates about30-40% production of GlcNAcMan₃GlcNA₂. The notation with (++++)indicates about 40-50% production of GlcNAcMan₃GlcNA₂. The notation with(+++++) indicates greater than 50% production of GlcNAcMan₃GlcNA₂. Thenotation (NG) indicates that no apparent glycans detected from anycolonies transformed with the fusion construct. TABLE 11 CatalyticDomains D. melanogaster D. melanogaster human D. melanogaster C. elegansmannosidase 1I mannosidase 1I mannosidase mannosidase 1I mannosidaseLeaders Δ48 Δ99 1I Δ48 Δ74 1I Δ108 Gls1-s ++ − + +++++ +++++ Gls1-m + −− − ++ Gls1-l − − − ++ ++ Mns1-s ++ − − ++++ ++++ Mns1-m ++ − − ++++++++ Mns1-l − − − ++ + S.Sec-s ++ − − ++++ ++++ S.Sec-m ++ − + ++++ ++++S.Sec-l + − + ++++ ++++ P.Sec-s ++ − + ++++ ++++ P.Sec-m − − + ++++ ++P.Och-s − − − ++ ++ P.Och-m − − − +++ +++ P.Och-l − − − ++ ++ Mnn9-s ++− − ++++ +++ Mnn9-m − − − − − Mnn9-l − − − ++ ++ Van1-s + − − ++++++++++ Van1-m − − − +++++ +++++ Van1-l − − − +++++ ++++ Anp1-s − − −+++++ ++++ Anp1-m − − − +++++ ++ Anp1-l − − − ++++ +++ Hoc1-s − − − +++++++ Hoc1-m − − − ++++ +++++ Hoc1-l + − − +++++ ++ Mnn10-s − − − + +++++Mnn10-m − − − ++++ +++++ Mnn10-l − − − +++ +++++ Mnn11-s + − − ++++++++++ Mnn11-m + − + − +++++ Mnn11-l − − − ++ ++ Mnt1-s + − − +++ +++++Mnt1-m + + − ++++ +++++ Mnt1-l + + − +++ +++++ D2-s − − − ++ +++++D2-m + − − +++ +++++ D2-l − − − + ++ D9-s − − − +++ +++ D9-m − − − ++++++ D9-l − − − + ++ J3-s + − + − ++ J3-m − − − +++++ ++++ J3-l − −− + + Ktr1-s + − − +++++ +++ Ktr1-l − − − + ++ Ktr2-s + − − +++++ +++++Ktr2-m + − − ++ +++ Ktr2-l + − − + ++ Gnt1-s + + − ++++ ++++ Gnt1-m −− + +++++ +++++ Gnt1-l − + − ++++ ++ Mnn2-s + + + ++++ ++++ Mnn2-m + + −++++ ++++ Mnn2-l + + − ++++ ++++ Mnn5-s − + − +++ ++++ Mnn5-m − + −+++++ +++++ Mnn5-l − + − ++ +++ Yur1-s − − − ++ +++ Yur1-m − − − + +++Yur1-l − − − − ++++ Mnn1-s − + + +++++ +++++ Mnn1-m + − + +++++ +++++Mnn1-l + − + +++++ +++ Mnn6-s − − + ++++ +++ Mnn6-m + − + +++++ ++++Mnn6-l − − − ++ +++

EXAMPLE 15 Generation of GnTII Expression Constructs

[0767] The construction of a GnTI expression vector (pNA15) containing ahuman GnTI gene fused with the N-terminal part of S. cerevisiae MNN9gene was described previously (Choi et al. Proc Natl Acad Sci U S A.2003 Apr. 29;100(9):5022-7). In a similar fashion, the rat GnTII genewas cloned. The rat GnTII gene (GenBank accession number U21662) was PCRamplified using Takara EX Taq™ polymerase (Panvera) from rat liver cDNAlibrary (Clontech) with RAT1 (5′-TTCCTCACTGCAGTCTTCTATAACT-3′) and RAT2(5′-TGGAGACCATGAGGTTCCGCATCTAC-3′) primers. The PCR product was thencloned into pCR2.1-TOPO vector (Invitrogen) and sequenced. Using thisvector as a template, the AscI-PacI fragment of GnTII, encodingamino-acids 88-443, was amplified with Pfu Turbo polymerase (Stratagene)and primers, RAT44 and RAT11 (5′-TTGGCGCGCCTCCCT AGTGTACCAGTTGAACTTTG-3′and 5′-GATTAATTAACTCACTGCAGTCTTCTATAACT -3′ respectively, introducedAscI and PacI restriction sites are underlined). Following confirmationby sequencing, the catalytic domain of rat GnTII was than cloneddownstream of the PMA1 promoter as a AscI-PacI fragment in pBP124. Inthe final step, the gene fragment encoding the S. cerevisiae Mnn2localization signal was cloned from pJN281 as a NotI-AscI fragment togenerate an in-frame fusion with the catalytic domain of GnTII, togenerate plasmid pTC53.

EXAMPLE 16 Reporter Protein Expression, Purification and Release ofN-linked Glycans

[0768] The K3 domain, under the control of the alcohol oxidase 1 (AOX1)promoter, was used as a model glycoprotein and was purified using thehexa-histidine tag as reported in Choi et al. Proc Natl Acad Sci U S A.2003 Apr. 29;100(9):5022-7). The glycans were released and separatedfrom the glycoproteins by a modification of a previously reported method(Papac et al. A. J. S. (1998) Glycobiology 8, 445-454). After theproteins were reduced and carboxymethylated, and the membranes blocked,the wells were washed three times with water. The protein wasdeglycosylated by the addition of 30 μl of 10 mM NH₄HCO₃ pH 8.3containing one milliunit of N-glycanase (Glyko). After incubation for 16hr at 37° C., the solution containing the glycans was removed bycentrifugation and evaporated to dryness.

[0769] Protein Purification

[0770] Kringle 3 was purified using a 96-well format on a Beckman BioMek2000 sample-handling robot (Beckman/Coulter Ranch Cucamonga, Calif.).Kringle 3 was purified from expression media using a C-terminalhexa-histidine tag. The robotic purification was an adaptation of theprotocol provided by Novagen for their HisBind resin. Briefly, a 150 uL(μL) settled volume of resin was poured into the wells of a 96-welllysate-binding plate, washed with 3 volumes of water and charged with 5volumes of 50 mM NiSO4 and washed with 3 volumes of binding buffer (5 mMimidazole, 0.5M NaCl, 20 mM Tris-HCL pH7.9). The protein expressionmedia was diluted 3:2, media/PBS (60 mM PO4, 16 mM KCl, 822 mM NaClpH7.4) and loaded onto the columns. After draining, the columns werewashed with 10 volumes of binding buffer and 6 volumes of wash buffer(30 mM imidazole, 0.5M NaCl, 20 mM Tris-HCl pH7.9) and the protein waseluted with 6 volumes of elution buffer (1M imidazole, 0.5M NaCl, 20 mMTris-HCl pH7.9). The eluted glycoproteins were evaporated to dryness bylyophilyzation.

[0771] Release of N-linked Glycans

[0772] The glycans were released and separated from the glycoproteins bya modification of a previously reported method (Papac, et al. A. J. S.(1998) Glycobiology 8, 445-454). The wells of a 96-well MultiScreen IP(Immobilon-P membrane) plate (Millipore) were wetted with 100 uL ofmethanol, washed with 3×150 uL of water and 50 uL of RCM buffer (8Murea, 360 mM Tris, 3.2 mM EDTA pH8.6), draining with gentle vacuum aftereach addition. The dried protein samples were dissolved in 30 uL of RCMbuffer and transferred to the wells containing 10 uL of RCM buffer. Thewells were drained and washed twice with RCM buffer. The proteins werereduced by addition of 60 uL of 0.1M DTT in RCM buffer for lhr at 37° C.The wells were washed three times with 300 uL of water andcarboxymethylated by addition of 60 uL of 0.1M iodoacetic acid for 30min in the dark at room temperature. The wells were again washed threetimes with water and the membranes blocked by the addition of 100 uL of1% PVP 360 in water for 1 hr at room temperature. The wells were drainedand washed three times with 300 uL of water and deglycosylated by theaddition of 30 uL of 10 mM NH₄HCO₃ pH 8.3 containing one milliunit ofN-glycanase (Glyko). After 16 hours at 37° C., the solution containingthe glycans was removed by centrifugation and evaporated to dryness.

[0773] MALDI/Time-of-Flight (TOF) Mass Spectrometry.

[0774] Molecular weights of the glycans were determined using a VoyagerDE PRO linear MALDI-TOF (Applied Biosciences) mass spectrometer usingdelayed extraction. The dried glycans from each well were dissolved in15 μl of water and 0.5 μl was spotted on stainless steel sample platesand mixed with 0.5 μl of S-DHB matrix (9 mg/ml of dihydroxybenzoic acid,1 mg/nl of 5-methoxysalicilic acid in 1:1 water/acetonitrile 0.1% TFA)and allowed to dry. Ions were generated by irradiation with a pulsednitrogen laser (337 nm) with a 4 ns pulse time. The instrument wasoperated in the delayed extraction mode with a 125 ns delay and anaccelerating voltage of 20 kV. The grid voltage was 93.00%, guide wirevoltage was 0.1%, the internal pressure was less than 5×10⁻⁷ torr, andthe low mass gate was 875 Da. Spectra were generated from the sum of100-200 laser pulses and acquired with a 500 MHz digitizer. Man₅GlcNAc₂oligosaccharide was used as an external molecular weight standard. Allspectra were generated with the instrument in the positive ion mode.

[0775] Miscellaneous:

[0776] Proteins were separated by SDS/PAGE according to Laemmli (Laemmli1970).

EXAMPLE 17 Generation of Yeast Strain YSH-1 (Δoch1, α1,2-mannosidase,GnTI)

[0777] The previously reported P. pastoris strain BK64 (Choi et al. ProcNatl Acad Sci USA. 2003 Apr. 29;100(9):5022-7), a triple auxotroph (ADE,ARG, HIS) possessing the OCH1 knock-out and expressing the kringle 3domain (K3) of human plasminogen, was used as the host strain. BK64 wastransformed with the plasmid pPB 103 linearized with the restrictionenzyme EcoNI to introduce the K.lactis UDP-N-acetylglucosaminetransporter into the host cell, thus creating the strain PBP-1. Themouse MnsI was introduced into this strain by transformation with theplasmid pFB8 linearized with the restriction enzyme EcoN1, generatingstrain PBP-2. K3 glycan analysis from proteins isolated from strainPBP-2 demonstrated that the primary glycoform present was Man₅GlcNAc₂.

[0778] PBP-2 was subsequently transformed with the human GnTI plasmidpNA15 linearized with the restriction enzyme AatII, generating thestrain PBP-3. Analysis of the K3 glycoformns produced in strain PBP-3demonstrated that the hybrid glycan GlcNAcMan₅GlcNAc₂ was thepredominant structure. To recover the URA3 marker from PBP-3, thisstrain was grown in YPD prior to selection on minimal media containing5-Fluoroorotic (5-FOA, BioVectra) and uracil (Boeke et al., Mol. Gen.Genet. 197:345-346 (1984)). The recovered Ura-minus strain producingGlcNAcMan₅GlcNAc₂ glycoforms was designated YSH-1. The N-glycan profilefrom strain YSH-1 is shown in FIG. 13 and displays a predominant peak at1465 m/z corresponding to the mass of GlcNAcMan₅GlcNAc₂ [d].

EXAMPLE 18 Generation of Yeast Strain YSH-37 (P. pastoris ExpressingMannosidase II)

[0779] YSH-1 (Example 17) was transformed with the D. melanogasiermannosidase IIΔ74/S. cerevisiae MNN2(s) plasmid (pKD53) linearized withthe restriction enzyme ApaI, generating strain YSH-37. Analysis of theK3 glycan structures produced in strain YSH-37 (FIG. 14) demonstratedthat the predominant glycoform at 1140 m/z corresponds to the mass ofGlcNAcMan₃GlcNAc₂ [b] and other glycoforms GlcNAcMan₄GlcNAc₂ [c] at 1303m/z and GlcNAcMan₅GlcNAc₂ [d] at 1465 m/z.

EXAMPLE 19 Generation of Yeast Strain YSH-44

[0780] Strain YSH-37 (Example 18) was transformed with a plasmidencoding a rat GnT II/MNN2 (s) leader, pTC53, linearized with therestriction enzyme EcoRI. The resulting strain, YSH-44, produced a K3N-glycan having a single glycoform at 1356 m/z, corresponding to themass of GlcNAc₂Man₃GlcNAc₂ [x], by positive mode MALDI-TOF massspectrometry (FIG. 15).

[0781] β-N-acetylhexosaminidase Digestion

[0782] The glycans from YSH-44 were released and separated from theglycoproteins by a modification of a previously reported method (Papac,et al. A. J. S. (1998) Glycobiology 8, 445-454). After the proteins werereduced and carboxymethylated and the membranes blocked, the wells werewashed three time with water. The protein was deglycosylated by theaddition of 30 μl of 10 mM NH₄HCO₃ pH 8.3 containing one milliunit ofN-glycanase (Glyko, Novato, Calif.). After a 16 hr digestion at 37° C.,the solution containing the glycans was removed by centrifugation andevaporated to dryness. The glycans were then dried in aSC210A speed vac(Thermo Savant, Halbrook, N.Y.). The dried glycans were put in 50 mMNH₄Ac pH 5.0 at 37° C. overnight and 1 mU of hexos (Glyko, Novato,Calif.) was added. The glycans were analyzed and shown to contain asingle glycan shown in FIG. 16 at 933 m/z corresponding to the mass ofMan₃GlcNAc₂ [a].

EXAMPLE 20 Generation of a Yeast Strain with No Apparent Mannosidase IIActivity

[0783] YSH-1 was transfoined with a plasmid encoding a D. melanogastermannosidase IIΔ74/S. cerevisiae MNN9(m), plasmid pKD16, linearized withthe restriction enzyme EcoRI. The resulting strain produced a singleglycoform at 1464 m/z corresponding to the mass of Man₅GlcNAc₂ [d] bypositive mode MALDI-TOF mass spectrometry (FIG. 18). This strain thusexpressed no apparent mannosidase II activity from the D. melanogastermannosidase IIΔ74/S. cerevisiae MNS1(1) fusion contruct, at least withrespect to glycosylation of the K3 reporter glycoprotein.

EXAMPLE 21 Generation of a Yeast Strain having Mannosidase II Activity

[0784] YSH-1 was transformed with a plasmid encoding a D. melanogastermannosidase IIΔ74/S. cerevisiae MNS1(1), plasmid (pKD6), linearized withthe restriction enzyme EcoRI. The N-glycan profile of K3 glycoproteinexpressed in the resulting strain (FIG. 19) exhibited a predominant peakat 1464 m/z corresponding to the mass of Man₅GlcNAc₂ [d] and other peakscorresponding to GlcNAcMan₃GlcNAc₂ [b] at 1139 m/z and GlcNAcMan₄GlcNAc₂[c] at 1302 m/z. The resulting yeast strain thus expressed somedetectable mannosidase II activity from the D. melanogaster mannosidaseIIΔ74/S. cerevisiae MNS1(1) fusion contruct.

EXAMPLE 22 Generation of Yeast Strain YSH-27 having Mannosidase IIActivity

[0785] YSH-1 was transformed with D. melanogaster mannosidase IIΔ74/S.cerevisiae GLS1(s) plasmid (pKD1), linearized with the restrictionenzyme EcoRI. The N-glycan profile of K3 glycoprotein expressed in theresulting strain, YSH-27, exhibited a predominant peak at 1139 m/zcorresponding to the mass of GlcNAcMan₃GlcNAc₂ [b] (FIG. 20). Theresulting strain YSH-27 thus expressed significant levels of mannosidaseII activity from the D. melanogaster mannosidase IIΔ74/S. cerevisiaeGLS1(s) fusion contruct.

EXAMPLE 23 Generation of Yeast Strain YSH-74 (Low Mannosidase IIActivity)

[0786] YSH-1 was transfonned with D. melanogaster mannosidase IIΔ74/S.cerevisiae MNS1(m) plasmid (pKD5), linearized with the restrictionenzyme EcoRI. The N-glycan profile of K3 glycoprotein expressed in theresulting strain, YSH-74, exhibited a predominant peak at 1140 m/zcorresponding to the mass of GlcNAcMan₃GlcNAc₂ [b] and other peakscorresponding to GlcNAcMan₄GlcNAc₂ [c] at 1302 m/z and GlcNAcMan₅GlcNAc₂[d] at 1464 m/z (FIG. 21). The resulting strain YSH-74 expressedmediocre levels of mannosidase II activity from the D. melanogastermannosidase IIΔ74/S. cerevisiae MNS1(m) fusion contruct, at least withrespect to glycosylation of the K3 reporter glycoprotein. The glycansfrom YSH-74 were analyzed further by digestion with A. saitoi α-1,2mannosidase (Glyko, Novato, Calif.), which resulted in glycansexhibiting a predominant peak at 1141 m/z corresponding to the mass ofGlcNAcMan₃GlcNAc₂ [b] (FIG. 22).

EXAMPLE 24 Mannosidase Assays

[0787] Fluorescently-labeled Man₈GlcNAc₂ (0.5 μg) was added to 20 μL ofsupernatant and incubated for 30 hours at room temperature. Afterincubation, the sample was analyzed by HPLC with an Econosil NH₂ 4.6×250mm, 5 micron bead, amino-bound silica column (Altech, Avondale, Pa.).The flow rate was 1.0 ml/min for 40 min and the column was maintained to30° C. After eluting isocratically (68% A:32% B) for 3 min, a linearsolvent gradient (68% A:32% B to 40% A:60% B) was employed over 27 minto elute the glycans (Turco, S. J. (1981) Anal. Biochem. 118, 278-283).Solvent A (acetonitrile) and solvent B was an aqueous solution ofammonium formate, 50 mM, pH 4.5. The column was equilibrated withsolvent (68% A:32% B) for 20 min between rpins.

EXAMPLE 25 In vitro Galactose Transfer

[0788] N-linked glycan GlcNAc₂Man₃GlcNAc₂ obtained from strain YSH-44was used as the substrate for galactose transfer. Twenty mg of thisglycan were incubated with 75 mg UDP-Gal and 10 to 50 mUβ-1,4-galactosyltranferase (Bovine milk, Calbiochem) in 50 mM NH₄HCO₃ ,1 mM MnCl₂, pH7.5 at 37° C. for 16-20 hr. FIG. 17 shows a positive modeMALDI-TOF mass spectrometry displaying a uniform peak at 1665 m/zcorresponding to the mass of Gal₂GlcNAc₂Man₃GlcNAc₂. The negativecontrol, minus galactosyltransferase, was carried out as described aboveand showed no transfer of galactose to the substrate GlcNAc₂Man₃GlcNAc₂.

EXAMPLE 26 Introduction of a Class III Mannosidase into Lower Eukaryotes

[0789] A cDNA encoding a class III mannosidase (Jarvis et al.Glycobiology 1997 7:113-127) from insect Sf9 cells was amplified usingprimers specific for the 5′ and 3′ termini. Subsequently, the cDNA wassubdloned into a yeast integration plasmid to investigate the effect ofthis protein on the N-glycosylation pattern of a secreted reporterprotein. A number of truncated products of were produced to generate alibrary of class III mannosidase constructs with different targetingleader fragments, as described, e.g., in Example 14. In addition tobeing expressed alone in a desired host strain, resulting fusionproteins are expressed in combination with other glycosylation modifyingenzymes to enhance the production of a desired N-glycan structure.

[0790] Although the Sf9 nannosidase is the only cloned member of thisclass III to date, genes and ESTs that show significant homology to thisORF, and in particular the catalytic domain (residues 273 to 2241 of theORF). A library of class III mannosidases that possess a range oftemperature and pH optima is generated. In turn, this will enable theselection of one or more class III mannosidase fusion constructs thatperform optimally in modifying the glycosylation pattern of a selectedreporter protein to produce a desired N-glycan structure when expressedin a desired host strain such as yeast and filamentous fungi.

1 119 1 1968 DNA Mus musculus CDS (1)..(1965) 1 atg ccc gtg ggg ggc ctgttg ccg ctc ttc agt agc cct ggg ggc ggc 48 Met Pro Val Gly Gly Leu LeuPro Leu Phe Ser Ser Pro Gly Gly Gly 1 5 10 15 ggc ctg ggc agt ggc ctgggc ggg ggg ctt ggc ggc ggg agg aag ggg 96 Gly Leu Gly Ser Gly Leu GlyGly Gly Leu Gly Gly Gly Arg Lys Gly 20 25 30 tct ggc ccc gct gcc ttc cgcctc acc gag aag ttc gtg ctg ctg ctg 144 Ser Gly Pro Ala Ala Phe Arg LeuThr Glu Lys Phe Val Leu Leu Leu 35 40 45 gtg ttc agc gcc ttc atc acg ctctgc ttc ggg gca atc ttc ttc ctg 192 Val Phe Ser Ala Phe Ile Thr Leu CysPhe Gly Ala Ile Phe Phe Leu 50 55 60 cct gac tcc tcc aag ctg ctc agc ggggtc ctg ttc cac tcc aac cct 240 Pro Asp Ser Ser Lys Leu Leu Ser Gly ValLeu Phe His Ser Asn Pro 65 70 75 80 gcc ttg cag ccg ccg gcg gag cac aagccc ggg ctc ggg gcg cgt gcg 288 Ala Leu Gln Pro Pro Ala Glu His Lys ProGly Leu Gly Ala Arg Ala 85 90 95 gag gat gcc gcc gag ggg aga gtc cgg caccgc gag gaa ggc gcg cct 336 Glu Asp Ala Ala Glu Gly Arg Val Arg His ArgGlu Glu Gly Ala Pro 100 105 110 ggg gac cct gga gct gga ctg gaa gac aactta gcc agg atc cgc gaa 384 Gly Asp Pro Gly Ala Gly Leu Glu Asp Asn LeuAla Arg Ile Arg Glu 115 120 125 aac cac gag cgg gct ctc agg gaa gcc aaggag acc ctg cag aag ctg 432 Asn His Glu Arg Ala Leu Arg Glu Ala Lys GluThr Leu Gln Lys Leu 130 135 140 ccg gag gag atc caa aga gac att ctg ctggag aag gaa aag gtg gcc 480 Pro Glu Glu Ile Gln Arg Asp Ile Leu Leu GluLys Glu Lys Val Ala 145 150 155 160 cag gac cag ctg cgt gac aag gat ctgttt agg ggc ttg ccc aag gtg 528 Gln Asp Gln Leu Arg Asp Lys Asp Leu PheArg Gly Leu Pro Lys Val 165 170 175 gac ttc ctg ccc ccc gtc ggg gta gagaac cgg gag ccc gct gac gcc 576 Asp Phe Leu Pro Pro Val Gly Val Glu AsnArg Glu Pro Ala Asp Ala 180 185 190 acc atc cgt gag aag agg gca aag atcaaa gag atg atg acc cat gct 624 Thr Ile Arg Glu Lys Arg Ala Lys Ile LysGlu Met Met Thr His Ala 195 200 205 tgg aat aat tat aaa cgc tat gcg tggggc ttg aac gaa ctg aaa cct 672 Trp Asn Asn Tyr Lys Arg Tyr Ala Trp GlyLeu Asn Glu Leu Lys Pro 210 215 220 ata tca aaa gaa ggc cat tca agc agtttg ttt ggc aac atc aaa gga 720 Ile Ser Lys Glu Gly His Ser Ser Ser LeuPhe Gly Asn Ile Lys Gly 225 230 235 240 gct aca ata gta gat gcc ctg gatacc ctt ttc att atg ggc atg aag 768 Ala Thr Ile Val Asp Ala Leu Asp ThrLeu Phe Ile Met Gly Met Lys 245 250 255 act gaa ttt caa gaa gct aaa tcgtgg att aaa aaa tat tta gat ttt 816 Thr Glu Phe Gln Glu Ala Lys Ser TrpIle Lys Lys Tyr Leu Asp Phe 260 265 270 aat gtg aat gct gaa gtt tct gttttt gaa gtc aac ata cgc ttc gtc 864 Asn Val Asn Ala Glu Val Ser Val PheGlu Val Asn Ile Arg Phe Val 275 280 285 ggt gga ctg ctg tca gcc tac tatttg tcc gga gag gag ata ttt cga 912 Gly Gly Leu Leu Ser Ala Tyr Tyr LeuSer Gly Glu Glu Ile Phe Arg 290 295 300 aag aaa gca gtg gaa ctt ggg gtaaaa ttg cta cct gca ttt cat act 960 Lys Lys Ala Val Glu Leu Gly Val LysLeu Leu Pro Ala Phe His Thr 305 310 315 320 ccc tct gga ata cct tgg gcattg ctg aat atg aaa agt ggg atc ggg 1008 Pro Ser Gly Ile Pro Trp Ala LeuLeu Asn Met Lys Ser Gly Ile Gly 325 330 335 cgg aac tgg ccc tgg gcc tctgga ggc agc agt atc ctg gcc gaa ttt 1056 Arg Asn Trp Pro Trp Ala Ser GlyGly Ser Ser Ile Leu Ala Glu Phe 340 345 350 gga act ctg cat tta gag tttatg cac ttg tcc cac tta tca gga gac 1104 Gly Thr Leu His Leu Glu Phe MetHis Leu Ser His Leu Ser Gly Asp 355 360 365 cca gtc ttt gcc gaa aag gttatg aaa att cga aca gtg ttg aac aaa 1152 Pro Val Phe Ala Glu Lys Val MetLys Ile Arg Thr Val Leu Asn Lys 370 375 380 ctg gac aaa cca gaa ggc ctttat cct aac tat ctg aac ccc agt agt 1200 Leu Asp Lys Pro Glu Gly Leu TyrPro Asn Tyr Leu Asn Pro Ser Ser 385 390 395 400 gga cag tgg ggt caa catcat gtg tcg gtt gga gga ctt gga gac agc 1248 Gly Gln Trp Gly Gln His HisVal Ser Val Gly Gly Leu Gly Asp Ser 405 410 415 ttt tat gaa tat ttg cttaag gcg tgg tta atg tct gac aag aca gat 1296 Phe Tyr Glu Tyr Leu Leu LysAla Trp Leu Met Ser Asp Lys Thr Asp 420 425 430 ctc gaa gcc aag aag atgtat ttt gat gct gtt cag gcc atc gag act 1344 Leu Glu Ala Lys Lys Met TyrPhe Asp Ala Val Gln Ala Ile Glu Thr 435 440 445 cac ttg atc cgc aag tcaagt ggg gga cta acg tac atc gca gag tgg 1392 His Leu Ile Arg Lys Ser SerGly Gly Leu Thr Tyr Ile Ala Glu Trp 450 455 460 aag ggg ggc ctc ctg gaacac aag atg ggc cac ctg acg tgc ttt gca 1440 Lys Gly Gly Leu Leu Glu HisLys Met Gly His Leu Thr Cys Phe Ala 465 470 475 480 gga ggc atg ttt gcactt ggg gca gat gga gct ccg gaa gcc cgg gcc 1488 Gly Gly Met Phe Ala LeuGly Ala Asp Gly Ala Pro Glu Ala Arg Ala 485 490 495 caa cac tac ctt gaactc gga gct gaa att gcc cgc act tgt cat gaa 1536 Gln His Tyr Leu Glu LeuGly Ala Glu Ile Ala Arg Thr Cys His Glu 500 505 510 tct tat aat cgt acatat gtg aag ttg gga ccg gaa gcg ttt cga ttt 1584 Ser Tyr Asn Arg Thr TyrVal Lys Leu Gly Pro Glu Ala Phe Arg Phe 515 520 525 gat ggc ggt gtg gaagct att gcc acg agg caa aat gaa aag tat tac 1632 Asp Gly Gly Val Glu AlaIle Ala Thr Arg Gln Asn Glu Lys Tyr Tyr 530 535 540 atc tta cgg ccc gaggtc atc gag aca tac atg tac atg tgg cga ctg 1680 Ile Leu Arg Pro Glu ValIle Glu Thr Tyr Met Tyr Met Trp Arg Leu 545 550 555 560 act cac gac cccaag tac agg acc tgg gcc tgg gaa gcc gtg gag gct 1728 Thr His Asp Pro LysTyr Arg Thr Trp Ala Trp Glu Ala Val Glu Ala 565 570 575 cta gaa agt cactgc aga gtg aac gga ggc tac tca ggc tta cgg gat 1776 Leu Glu Ser His CysArg Val Asn Gly Gly Tyr Ser Gly Leu Arg Asp 580 585 590 gtt tac att gcccgt gag agt tat gac gat gtc cag caa agt ttc ttc 1824 Val Tyr Ile Ala ArgGlu Ser Tyr Asp Asp Val Gln Gln Ser Phe Phe 595 600 605 ctg gca gag acactg aag tat ttg tac ttg ata ttt tcc gat gat gac 1872 Leu Ala Glu Thr LeuLys Tyr Leu Tyr Leu Ile Phe Ser Asp Asp Asp 610 615 620 ctt ctt cca ctagaa cac tgg atc ttc aac acc gag gct cat cct ttc 1920 Leu Leu Pro Leu GluHis Trp Ile Phe Asn Thr Glu Ala His Pro Phe 625 630 635 640 cct ata ctccgt gaa cag aag aag gaa att gat ggc aaa gag aaa tga 1968 Pro Ile Leu ArgGlu Gln Lys Lys Glu Ile Asp Gly Lys Glu Lys 645 650 655 2 655 PRT Musmusculus 2 Met Pro Val Gly Gly Leu Leu Pro Leu Phe Ser Ser Pro Gly GlyGly 1 5 10 15 Gly Leu Gly Ser Gly Leu Gly Gly Gly Leu Gly Gly Gly ArgLys Gly 20 25 30 Ser Gly Pro Ala Ala Phe Arg Leu Thr Glu Lys Phe Val LeuLeu Leu 35 40 45 Val Phe Ser Ala Phe Ile Thr Leu Cys Phe Gly Ala Ile PhePhe Leu 50 55 60 Pro Asp Ser Ser Lys Leu Leu Ser Gly Val Leu Phe His SerAsn Pro 65 70 75 80 Ala Leu Gln Pro Pro Ala Glu His Lys Pro Gly Leu GlyAla Arg Ala 85 90 95 Glu Asp Ala Ala Glu Gly Arg Val Arg His Arg Glu GluGly Ala Pro 100 105 110 Gly Asp Pro Gly Ala Gly Leu Glu Asp Asn Leu AlaArg Ile Arg Glu 115 120 125 Asn His Glu Arg Ala Leu Arg Glu Ala Lys GluThr Leu Gln Lys Leu 130 135 140 Pro Glu Glu Ile Gln Arg Asp Ile Leu LeuGlu Lys Glu Lys Val Ala 145 150 155 160 Gln Asp Gln Leu Arg Asp Lys AspLeu Phe Arg Gly Leu Pro Lys Val 165 170 175 Asp Phe Leu Pro Pro Val GlyVal Glu Asn Arg Glu Pro Ala Asp Ala 180 185 190 Thr Ile Arg Glu Lys ArgAla Lys Ile Lys Glu Met Met Thr His Ala 195 200 205 Trp Asn Asn Tyr LysArg Tyr Ala Trp Gly Leu Asn Glu Leu Lys Pro 210 215 220 Ile Ser Lys GluGly His Ser Ser Ser Leu Phe Gly Asn Ile Lys Gly 225 230 235 240 Ala ThrIle Val Asp Ala Leu Asp Thr Leu Phe Ile Met Gly Met Lys 245 250 255 ThrGlu Phe Gln Glu Ala Lys Ser Trp Ile Lys Lys Tyr Leu Asp Phe 260 265 270Asn Val Asn Ala Glu Val Ser Val Phe Glu Val Asn Ile Arg Phe Val 275 280285 Gly Gly Leu Leu Ser Ala Tyr Tyr Leu Ser Gly Glu Glu Ile Phe Arg 290295 300 Lys Lys Ala Val Glu Leu Gly Val Lys Leu Leu Pro Ala Phe His Thr305 310 315 320 Pro Ser Gly Ile Pro Trp Ala Leu Leu Asn Met Lys Ser GlyIle Gly 325 330 335 Arg Asn Trp Pro Trp Ala Ser Gly Gly Ser Ser Ile LeuAla Glu Phe 340 345 350 Gly Thr Leu His Leu Glu Phe Met His Leu Ser HisLeu Ser Gly Asp 355 360 365 Pro Val Phe Ala Glu Lys Val Met Lys Ile ArgThr Val Leu Asn Lys 370 375 380 Leu Asp Lys Pro Glu Gly Leu Tyr Pro AsnTyr Leu Asn Pro Ser Ser 385 390 395 400 Gly Gln Trp Gly Gln His His ValSer Val Gly Gly Leu Gly Asp Ser 405 410 415 Phe Tyr Glu Tyr Leu Leu LysAla Trp Leu Met Ser Asp Lys Thr Asp 420 425 430 Leu Glu Ala Lys Lys MetTyr Phe Asp Ala Val Gln Ala Ile Glu Thr 435 440 445 His Leu Ile Arg LysSer Ser Gly Gly Leu Thr Tyr Ile Ala Glu Trp 450 455 460 Lys Gly Gly LeuLeu Glu His Lys Met Gly His Leu Thr Cys Phe Ala 465 470 475 480 Gly GlyMet Phe Ala Leu Gly Ala Asp Gly Ala Pro Glu Ala Arg Ala 485 490 495 GlnHis Tyr Leu Glu Leu Gly Ala Glu Ile Ala Arg Thr Cys His Glu 500 505 510Ser Tyr Asn Arg Thr Tyr Val Lys Leu Gly Pro Glu Ala Phe Arg Phe 515 520525 Asp Gly Gly Val Glu Ala Ile Ala Thr Arg Gln Asn Glu Lys Tyr Tyr 530535 540 Ile Leu Arg Pro Glu Val Ile Glu Thr Tyr Met Tyr Met Trp Arg Leu545 550 555 560 Thr His Asp Pro Lys Tyr Arg Thr Trp Ala Trp Glu Ala ValGlu Ala 565 570 575 Leu Glu Ser His Cys Arg Val Asn Gly Gly Tyr Ser GlyLeu Arg Asp 580 585 590 Val Tyr Ile Ala Arg Glu Ser Tyr Asp Asp Val GlnGln Ser Phe Phe 595 600 605 Leu Ala Glu Thr Leu Lys Tyr Leu Tyr Leu IlePhe Ser Asp Asp Asp 610 615 620 Leu Leu Pro Leu Glu His Trp Ile Phe AsnThr Glu Ala His Pro Phe 625 630 635 640 Pro Ile Leu Arg Glu Gln Lys LysGlu Ile Asp Gly Lys Glu Lys 645 650 655 3 26 DNA Kluyveromyces lactis 3ccagaagaat tcaattytgy cartgg 26 4 25 DNA Kluyveromyces lactismodified_base (17) a, c, g, t, other or unknown 4 cagtgaaaat acctggnccngtcca 25 5 23 PRT Unknown Organism Description of Unknown Organism Class2 mannosidase conserved amino acid sequence 5 Leu Lys Val Phe Val ValPro His Ser His Asn Asp Pro Gly Trp Ile 1 5 10 15 Gln Thr Phe Glu GluTyr Tyr 20 6 57 PRT Unknown Organism Description of Unknown OrganismClass 2 mannosidase conserved amino acid sequence 6 Glu Phe Val Thr GlyGly Trp Val Met Pro Asp Glu Ala Asn Ser Trp 1 5 10 15 Arg Asn Val LeuLeu Gln Leu Thr Glu Gly Gln Thr Trp Leu Lys Gln 20 25 30 Phe Met Asn ValThr Pro Thr Ala Ser Trp Ala Ile Asp Pro Phe Gly 35 40 45 His Ser Pro ThrMet Pro Tyr Ile Leu 50 55 7 33 PRT Unknown Organism Description ofUnknown Organism Class 2 mannosidase conserved amino acid sequence 7 HisMet Met Pro Phe Tyr Ser Tyr Asp Ile Pro His Thr Cys Gly Pro 1 5 10 15Asp Pro Arg Ile Cys Cys Gln Phe Asp Phe Arg Arg Met Pro Gly Gly 20 25 30Arg 8 28 PRT Unknown Organism Description of Unknown Organism Class 2mannosidase conserved amino acid sequence 8 Leu Leu Leu Asp Gln Tyr ArgLys Lys Ser Glu Leu Phe Arg Thr Asn 1 5 10 15 Val Leu Leu Ile Pro LeuGly Asp Asp Phe Arg Tyr 20 25 9 12 PRT Unknown Organism Description ofUnknown Organism Class 2 mannosidase conserved amino acid sequence 9 GlnPhe Gly Thr Leu Ser Asp Tyr Phe Asp Ala Leu 1 5 10 10 14 PRT UnknownOrganism Description of Unknown Organism Class 2 mannosidase conservedamino acid sequence 10 Leu Ser Gly Asp Phe Phe Thr Tyr Ala Asp Arg SerAsp His 1 5 10 11 20 PRT Unknown Organism Description of UnknownOrganism Class 2 mannosidase conserved amino acid sequence 11 Tyr TrpSer Gly Tyr Tyr Thr Ser Arg Pro Phe Tyr Arg Arg Met Asp 1 5 10 15 ArgVal Leu Glu 20 12 27 PRT Unknown Organism Description of UnknownOrganism Class 2 mannosidase conserved amino acid sequence 12 Ala ArgArg Glu Leu Gly Leu Phe Gln His His Asp Ala Ile Thr Gly 1 5 10 15 ThrAla Arg Asp His Val Val Val Asp Tyr Gly 20 25 13 11 PRT Unknown OrganismDescription of Unknown Organism Class 2 mannosidase conserved amino acidsequence 13 Gly Ala Tyr Leu Phe Leu Pro Asp Gly Glu Ala 1 5 10 14 14 PRTUnknown Organism Description of Unknown Organism Class 2 mannosidaseconserved amino acid sequence 14 Phe Tyr Thr Asp Leu Asn Gly Phe Gln MetGln Lys Arg Arg 1 5 10 15 66 PRT Unknown Organism Description of UnknownOrganism Class 2 mannosidase conserved amino acid sequence 15 Lys LeuPro Leu Gln Ala Asn Tyr Tyr Pro Met Pro Ser Met Ala Tyr 1 5 10 15 IleGln Asp Ala Asn Thr Arg Leu Thr Leu Leu Thr Gly Gln Pro Leu 20 25 30 GlyVal Ser Ser Leu Ala Ser Gly Gln Leu Glu Val Met Leu Asp Arg 35 40 45 ArgLeu Met Ser Asp Asp Asn Arg Gly Leu Gly Gln Gly Val Leu Asp 50 55 60 AsnLys 65 16 27 DNA Kluyveromyces lactis 16 tgccatcttt taggtccagg cccgttc27 17 27 DNA Kluyveromyces lactis 17 gatcccacga cgcatcgtat ttctttc 27 1821 DNA Artificial Sequence Description of Artificial Sequence Syntheticprimer 18 atggcgaagg cagatggcag t 21 19 21 DNA Artificial SequenceDescription of Artificial Sequence Synthetic primer 19 ttagtccttccaacttcctt c 21 20 21 DNA Artificial Sequence Description of ArtificialSequence Synthetic primer 20 actgccatct gccttcgcca t 21 21 22 DNAArtificial Sequence Description of Artificial Sequence Synthetic primer21 gtaatacgac tcactatagg gc 22 22 20 DNA Artificial Sequence Descriptionof Artificial Sequence Synthetic primer 22 aattaaccct cactaaaggg 20 2336 DNA Artificial Sequence Description of Artificial Sequence Syntheticprimer 23 atgcccgtgg ggggcctgtt gccgctcttc agtagc 36 24 40 DNAArtificial Sequence Description of Artificial Sequence Synthetic primer24 tcatttctct ttgccatcaa tttccttctt ctgttcacgg 40 25 44 DNA ArtificialSequence Description of Artificial Sequence Synthetic primer 25ggcgcgccga ctcctccaag ctgctcagcg gggtcctgtt ccac 44 26 50 DNA ArtificialSequence Description of Artificial Sequence Synthetic primer 26ccttaattaa tcatttctct ttgccatcaa tttccttctt ctgttcacgg 50 27 51 DNAArtificial Sequence Description of Artificial Sequence Synthetic primer27 ggcgagctcg gcctacccgg ccaaggctga gatcatttgt ccagcttcag a 51 28 55 DNAArtificial Sequence Description of Artificial Sequence Synthetic primer28 gcccacgtcg acggatccgt ttaaacatcg attggagagg ctgacaccgc tacta 55 29 55DNA Artificial Sequence Description of Artificial Sequence Syntheticprimer 29 cgggatccac tagtatttaa atcatatgtg cgagtgtaca actcttccca catgg55 30 55 DNA Artificial Sequence Description of Artificial SequenceSynthetic primer 30 ggacgcgtcg acggcctacc cggccgtacg aggaatttctcggatgactc ttttc 55 31 45 DNA Artificial Sequence Description ofArtificial Sequence Synthetic primer 31 cgggatccct cgagagatct tttttgtagaaatgtcttgg tgcct 45 32 63 DNA Artificial Sequence Description ofArtificial Sequence Synthetic primer 32 ggacatgcat gcactagtgc ggccgccacgtgatagttgt tcaattgatt gaaataggga 60 caa 63 33 52 DNA Artificial SequenceDescription of Artificial Sequence Synthetic primer 33 ccttgctagcttaattaacc gcggcacgtc cgacggcggc ccacgggtcc ca 52 34 61 DNA ArtificialSequence Description of Artificial Sequence Synthetic primer 34ggacatgcat gcggatccct taagagccgg cagcttgcaa attaaagcct tcgagcgtcc 60 c61 35 61 DNA Artificial Sequence Description of Artificial SequenceSynthetic primer 35 gaaccacgtc gacggccatt gcggccaaaa ccttttttcctattcaaaca caaggcattg 60 c 61 36 43 DNA Artificial Sequence Descriptionof Artificial Sequence Synthetic primer 36 ctccaatact agtcgaagattatcttctac ggtgcctgga ctc 43 37 53 DNA Artificial Sequence Descriptionof Artificial Sequence Synthetic primer 37 tggaaggttt aaacaaagctagagtaaaat agatatagcg agattagaga atg 53 38 50 DNA Artificial SequenceDescription of Artificial Sequence Synthetic primer 38 aagaattcggctggaaggcc ttgtaccttg atgtagttcc cgttttcatc 50 39 58 DNA ArtificialSequence Description of Artificial Sequence Synthetic primer 39gcccaagccg gccttaaggg atctcctgat gactgactca ctgataataa aaatacgg 58 40 59DNA Artificial Sequence Description of Artificial Sequence Syntheticprimer 40 gggcgcgtat ttaaatacta gtggatctat cgaatctaaa tgtaagttaaaatctctaa 59 41 39 DNA Artificial Sequence Description of ArtificialSequence Synthetic primer 41 ggccgcctgc agatttaaat gaattcggcg cgccttaat39 42 34 DNA Artificial Sequence Description of Artificial SequenceSynthetic primer 42 taaggcgcgc cgaattcatt taaatctgca gggc 34 43 29 DNAArtificial Sequence Description of Artificial Sequence Synthetic primer43 tggcaggcgc gcctcagtca gcgctctcg 29 44 29 DNA Artificial SequenceDescription of Artificial Sequence Synthetic primer 44 aggttaattaagtgctaatt ccagctagg 29 45 26 DNA Artificial Sequence Description ofArtificial Sequence Synthetic primer 45 ccagaagaat tcaattytgy cartgg 2646 25 DNA Artificial Sequence Description of Artificial SequenceSynthetic primer 46 cagtgaaaat acctggnccn gtcca 25 47 27 DNA ArtificialSequence Description of Artificial Sequence Synthetic primer 47tgccatcttt taggtccagg cccgttc 27 48 27 DNA Artificial SequenceDescription of Artificial Sequence Synthetic primer 48 gatcccacgacgcatcgtat ttctttc 27 49 3522 DNA Arabidopsis thaliana CDS (1)..(3519)49 atg ccg ttc tcc tcg tat atc ggc aac agc cgc cgt agc tcc acc ggc 48Met Pro Phe Ser Ser Tyr Ile Gly Asn Ser Arg Arg Ser Ser Thr Gly 1 5 1015 gga gga acc ggc ggt tgg ggc caa tct ctt ctt cca aca gcg tta tca 96Gly Gly Thr Gly Gly Trp Gly Gln Ser Leu Leu Pro Thr Ala Leu Ser 20 25 30aag tca aaa cta gcg atc aat cga aaa cca cga aaa cga act ctc gta 144 LysSer Lys Leu Ala Ile Asn Arg Lys Pro Arg Lys Arg Thr Leu Val 35 40 45 gtcaat ttc atc ttc gcc aac ttc ttc gtc atc gca ctc acc gtc tca 192 Val AsnPhe Ile Phe Ala Asn Phe Phe Val Ile Ala Leu Thr Val Ser 50 55 60 ctc ctcttc ttc ctc ctc act ctc ttc cac ttc ggc gta cca gga ccg 240 Leu Leu PhePhe Leu Leu Thr Leu Phe His Phe Gly Val Pro Gly Pro 65 70 75 80 atc tcctca cga ttc ctt acc tcc aga tcc aat cgg atc gtc aag cca 288 Ile Ser SerArg Phe Leu Thr Ser Arg Ser Asn Arg Ile Val Lys Pro 85 90 95 cgg aag aatatt aat cgc cga ccc tta aac gat tcc aat tca ggc gcc 336 Arg Lys Asn IleAsn Arg Arg Pro Leu Asn Asp Ser Asn Ser Gly Ala 100 105 110 gtc gtt gatatc aca act aaa gat cta tac gat agg att gag ttt ctt 384 Val Val Asp IleThr Thr Lys Asp Leu Tyr Asp Arg Ile Glu Phe Leu 115 120 125 gat aca gatggt ggt cca tgg aaa caa ggt tgg aga gtt acg tat aaa 432 Asp Thr Asp GlyGly Pro Trp Lys Gln Gly Trp Arg Val Thr Tyr Lys 130 135 140 gac gat gagtgg gag aaa gag aag ctc aaa atc ttc gtt gtt cct cat 480 Asp Asp Glu TrpGlu Lys Glu Lys Leu Lys Ile Phe Val Val Pro His 145 150 155 160 tct cataac gat cct ggt tgg aaa ttg act gta gag gag tat tat cag 528 Ser His AsnAsp Pro Gly Trp Lys Leu Thr Val Glu Glu Tyr Tyr Gln 165 170 175 aga caatcc aga cat att ctt gac acc att gtt gag act tta tct aag 576 Arg Gln SerArg His Ile Leu Asp Thr Ile Val Glu Thr Leu Ser Lys 180 185 190 gat tcaaga aga aag ttt ata tgg gag gag atg tca tat ctg gag aga 624 Asp Ser ArgArg Lys Phe Ile Trp Glu Glu Met Ser Tyr Leu Glu Arg 195 200 205 tgg tggaga gac gct tca cct aat aaa caa gaa gct ttg act aaa ttg 672 Trp Trp ArgAsp Ala Ser Pro Asn Lys Gln Glu Ala Leu Thr Lys Leu 210 215 220 gtt aaggat ggg cag cta gag att gtt gga ggt ggc tgg gtt atg aat 720 Val Lys AspGly Gln Leu Glu Ile Val Gly Gly Gly Trp Val Met Asn 225 230 235 240 gatgag gct aat tca cat tat ttt gcc ata att gaa cag ata gca gag 768 Asp GluAla Asn Ser His Tyr Phe Ala Ile Ile Glu Gln Ile Ala Glu 245 250 255 ggtaat atg tgg ctg aat gac aca att ggg gtt att cct aag aat tct 816 Gly AsnMet Trp Leu Asn Asp Thr Ile Gly Val Ile Pro Lys Asn Ser 260 265 270 tgggct ata gat ccc ttt ggc tat tca tca acc atg gct tat ctt ctc 864 Trp AlaIle Asp Pro Phe Gly Tyr Ser Ser Thr Met Ala Tyr Leu Leu 275 280 285 cggcgt atg ggt ttt gaa aac atg ctt att caa agg act cat tac gag 912 Arg ArgMet Gly Phe Glu Asn Met Leu Ile Gln Arg Thr His Tyr Glu 290 295 300 ctcaag aaa gac ctt gcc cag cat aag aat ctt gaa tat att tgg cgt 960 Leu LysLys Asp Leu Ala Gln His Lys Asn Leu Glu Tyr Ile Trp Arg 305 310 315 320cag agc tgg gat gct atg gaa acc aca gat atc ttt gtt cat atg atg 1008 GlnSer Trp Asp Ala Met Glu Thr Thr Asp Ile Phe Val His Met Met 325 330 335ccg ttt tat tca tac gat atc cca cac act tgt gga cca gag cct gca 1056 ProPhe Tyr Ser Tyr Asp Ile Pro His Thr Cys Gly Pro Glu Pro Ala 340 345 350att tgc tgt cag ttt gat ttc gct cgg atg cgg gga ttt aag tat gaa 1104 IleCys Cys Gln Phe Asp Phe Ala Arg Met Arg Gly Phe Lys Tyr Glu 355 360 365ctt tgt cca tgg gga aag cac cca gtg gag acc aca cta gaa aat gtg 1152 LeuCys Pro Trp Gly Lys His Pro Val Glu Thr Thr Leu Glu Asn Val 370 375 380cag gag agg gca tta aag ctt ctg gat caa tac agg aaa aaa tcc act 1200 GlnGlu Arg Ala Leu Lys Leu Leu Asp Gln Tyr Arg Lys Lys Ser Thr 385 390 395400 cta tat cga act aat aca ctt ctt ata cct ctt gga gat gat ttt agg 1248Leu Tyr Arg Thr Asn Thr Leu Leu Ile Pro Leu Gly Asp Asp Phe Arg 405 410415 tac att agt atc gat gaa gcc gag gct cag ttc cgt aac tac cag atg 1296Tyr Ile Ser Ile Asp Glu Ala Glu Ala Gln Phe Arg Asn Tyr Gln Met 420 425430 ttg ttt gat cac atc aac tct aat cct agt cta aac gca gaa gca aag 1344Leu Phe Asp His Ile Asn Ser Asn Pro Ser Leu Asn Ala Glu Ala Lys 435 440445 ttt ggt act ttg gag gat tat ttc aga aca gtc cga gaa gaa gca gac 1392Phe Gly Thr Leu Glu Asp Tyr Phe Arg Thr Val Arg Glu Glu Ala Asp 450 455460 aga gtg aat tat tct cgt cct ggt gag gtt ggc tct ggt cag gtt gtt 1440Arg Val Asn Tyr Ser Arg Pro Gly Glu Val Gly Ser Gly Gln Val Val 465 470475 480 ggt ttc cct tct ctg tca ggt gac ttc ttt aca tat gca gat agg caa1488 Gly Phe Pro Ser Leu Ser Gly Asp Phe Phe Thr Tyr Ala Asp Arg Gln 485490 495 caa gac tat tgg agt ggt tat tat gtt tca aga cct ttc ttc aaa gct1536 Gln Asp Tyr Trp Ser Gly Tyr Tyr Val Ser Arg Pro Phe Phe Lys Ala 500505 510 gtt gat cgt gtg ctc gag cat acc ctt cgt gga gct gag atc atg atg1584 Val Asp Arg Val Leu Glu His Thr Leu Arg Gly Ala Glu Ile Met Met 515520 525 tca ttt ctg cta ggt tat tgc cat cga att caa tgt gag aaa ttt cca1632 Ser Phe Leu Leu Gly Tyr Cys His Arg Ile Gln Cys Glu Lys Phe Pro 530535 540 aca agt ttt acg tat aag ttg act gct gca aga aga aat ctg gct ctt1680 Thr Ser Phe Thr Tyr Lys Leu Thr Ala Ala Arg Arg Asn Leu Ala Leu 545550 555 560 ttc cag cac cat gat ggg gta act gga act gct aag gat tat gtggta 1728 Phe Gln His His Asp Gly Val Thr Gly Thr Ala Lys Asp Tyr Val Val565 570 575 caa gat tac ggc acc cgg atg cat act tca ttg caa gac ctt cagatc 1776 Gln Asp Tyr Gly Thr Arg Met His Thr Ser Leu Gln Asp Leu Gln Ile580 585 590 ttt atg tct aaa gca atc gaa gtt ctt ctt ggg atc cgc cac gagaaa 1824 Phe Met Ser Lys Ala Ile Glu Val Leu Leu Gly Ile Arg His Glu Lys595 600 605 gaa aaa tct gat caa tcc cca tca ttt ttc gag gca gag caa atgaga 1872 Glu Lys Ser Asp Gln Ser Pro Ser Phe Phe Glu Ala Glu Gln Met Arg610 615 620 tca aag tat gat gct cgg cca gtt cac aag cca att gct gcc cgggaa 1920 Ser Lys Tyr Asp Ala Arg Pro Val His Lys Pro Ile Ala Ala Arg Glu625 630 635 640 gga aat tcg cac aca gtt ata ctc ttc aat cca tca gaa cagacg aga 1968 Gly Asn Ser His Thr Val Ile Leu Phe Asn Pro Ser Glu Gln ThrArg 645 650 655 gag gag gtg gtg acg gtt gtt gtt aac cgc gct gaa atc tcggtt ttg 2016 Glu Glu Val Val Thr Val Val Val Asn Arg Ala Glu Ile Ser ValLeu 660 665 670 gac tca aac tgg act tgt gtc cct agc caa att tct cct gaagtg cag 2064 Asp Ser Asn Trp Thr Cys Val Pro Ser Gln Ile Ser Pro Glu ValGln 675 680 685 cat gac gat acc aaa cta ttc acc ggc aga cat cgc ctt tactgg aaa 2112 His Asp Asp Thr Lys Leu Phe Thr Gly Arg His Arg Leu Tyr TrpLys 690 695 700 gct tcc atc cca gct ctt ggt ctg aga aca tat ttc att gctaat ggg 2160 Ala Ser Ile Pro Ala Leu Gly Leu Arg Thr Tyr Phe Ile Ala AsnGly 705 710 715 720 aat gtc gag tgt gag aaa gct act ccg tct aaa ctc aaatac gct tct 2208 Asn Val Glu Cys Glu Lys Ala Thr Pro Ser Lys Leu Lys TyrAla Ser 725 730 735 gag ttt gac cca ttt cct tgt cct cct cca tat tcc tgctcc aaa ctg 2256 Glu Phe Asp Pro Phe Pro Cys Pro Pro Pro Tyr Ser Cys SerLys Leu 740 745 750 gac aac gac gtt act gag atc cga aat gaa cat cag actctt gtg ttt 2304 Asp Asn Asp Val Thr Glu Ile Arg Asn Glu His Gln Thr LeuVal Phe 755 760 765 gat gtg aag aac gga tca ctg cgg aag ata gtc cat agaaac gga tca 2352 Asp Val Lys Asn Gly Ser Leu Arg Lys Ile Val His Arg AsnGly Ser 770 775 780 gag act gtt gtg gga gaa gag ata ggt atg tac tct agtcca gag agt 2400 Glu Thr Val Val Gly Glu Glu Ile Gly Met Tyr Ser Ser ProGlu Ser 785 790 795 800 gga gct tac ctg ttc aaa cca gat ggt gaa gct cagcca att gtt caa 2448 Gly Ala Tyr Leu Phe Lys Pro Asp Gly Glu Ala Gln ProIle Val Gln 805 810 815 cct gat gga cat gta gtc acc tct gag ggt ctg ctggtt caa gaa gtc 2496 Pro Asp Gly His Val Val Thr Ser Glu Gly Leu Leu ValGln Glu Val 820 825 830 ttc tct tac cct aaa acc aaa tgg gag aaa tca cccctc tct cag aaa 2544 Phe Ser Tyr Pro Lys Thr Lys Trp Glu Lys Ser Pro LeuSer Gln Lys 835 840 845 act cgt ctt tac act gga ggt aat acg ctt cag gatcaa gtg gtc gag 2592 Thr Arg Leu Tyr Thr Gly Gly Asn Thr Leu Gln Asp GlnVal Val Glu 850 855 860 ata gaa tat cat gtt gag ctt ctt ggt aat gat tttgat gac cgg gaa 2640 Ile Glu Tyr His Val Glu Leu Leu Gly Asn Asp Phe AspAsp Arg Glu 865 870 875 880 ttg att gtc cgg tac aag act gat gtt gac aacaag aag gtc ttc tat 2688 Leu Ile Val Arg Tyr Lys Thr Asp Val Asp Asn LysLys Val Phe Tyr 885 890 895 tca gat ctc aat ggt ttc caa atg agc agg agagaa act tat gat aag 2736 Ser Asp Leu Asn Gly Phe Gln Met Ser Arg Arg GluThr Tyr Asp Lys 900 905 910 atc cct ctt caa gga aac tac tac cca atg ccatct ctc gca ttt atc 2784 Ile Pro Leu Gln Gly Asn Tyr Tyr Pro Met Pro SerLeu Ala Phe Ile 915 920 925 caa gga tcc aat ggt cag aga ttc tcc gtg cactct cgt caa tct ctc 2832 Gln Gly Ser Asn Gly Gln Arg Phe Ser Val His SerArg Gln Ser Leu 930 935 940 ggt gtt gca agc ctc aaa gag ggt tgg ttg gagatt atg ctg gac aga 2880 Gly Val Ala Ser Leu Lys Glu Gly Trp Leu Glu IleMet Leu Asp Arg 945 950 955 960 cgg ttg gtt cgt gat gac gga cgg ggt ctaggg caa ggt gtg atg gat 2928 Arg Leu Val Arg Asp Asp Gly Arg Gly Leu GlyGln Gly Val Met Asp 965 970 975 aac cgc gca atg acc gtg gta ttt cac cttctt gcg gaa tct aac att 2976 Asn Arg Ala Met Thr Val Val Phe His Leu LeuAla Glu Ser Asn Ile 980 985 990 tct caa gca gac cct gct tcc aac act aacccg agg aac cct tcg ctt 3024 Ser Gln Ala Asp Pro Ala Ser Asn Thr Asn ProArg Asn Pro Ser Leu 995 1000 1005 ctc tct cac ctc ata ggt gct cac ttaaac tac ccc ata aac aca ttc 3072 Leu Ser His Leu Ile Gly Ala His Leu AsnTyr Pro Ile Asn Thr Phe 1010 1015 1020 att gcc aag aaa ccg caa gac atatct gtg cgt gtt cca caa tac ggt 3120 Ile Ala Lys Lys Pro Gln Asp Ile SerVal Arg Val Pro Gln Tyr Gly 1025 1030 1035 1040 tcc ttt gct cct tta gccaaa ccg tta cca tgt gac ctc cac att gta 3168 Ser Phe Ala Pro Leu Ala LysPro Leu Pro Cys Asp Leu His Ile Val 1045 1050 1055 aat ttc aag gtt cctcgt cca tcc aaa tac tct cag caa ttg gaa gaa 3216 Asn Phe Lys Val Pro ArgPro Ser Lys Tyr Ser Gln Gln Leu Glu Glu 1060 1065 1070 gac aag cca aggttc gct ctt atc ctc aat aga cga gct tgg gat tca 3264 Asp Lys Pro Arg PheAla Leu Ile Leu Asn Arg Arg Ala Trp Asp Ser 1075 1080 1085 gct tat tgccat aaa gga aga caa gta aac tgc aca agc atg gct aat 3312 Ala Tyr Cys HisLys Gly Arg Gln Val Asn Cys Thr Ser Met Ala Asn 1090 1095 1100 gaa ccagta aac ttt tcc gac atg ttc aaa gat ctt gca gct tca aag 3360 Glu Pro ValAsn Phe Ser Asp Met Phe Lys Asp Leu Ala Ala Ser Lys 1105 1110 1115 1120gta aaa cca act tca ctg aat ctc ttg caa gaa gat atg gag att ctt 3408 ValLys Pro Thr Ser Leu Asn Leu Leu Gln Glu Asp Met Glu Ile Leu 1125 11301135 ggg tac gat gac caa gag cta cct cga gat agt tca cag cca cgg gaa3456 Gly Tyr Asp Asp Gln Glu Leu Pro Arg Asp Ser Ser Gln Pro Arg Glu1140 1145 1150 gga cgt gtc tcg atc tct ccc atg gaa ata cga gct tat aagctt gaa 3504 Gly Arg Val Ser Ile Ser Pro Met Glu Ile Arg Ala Tyr Lys LeuGlu 1155 1160 1165 ctg cga cct cac aag tga 3522 Leu Arg Pro His Lys 117050 3432 DNA Caenorhabditis elegans CDS (1)..(3429) 50 atg gga aaa cgcaat ttc tat att atc cta tgt ttg gga gtc ttt ctc 48 Met Gly Lys Arg AsnPhe Tyr Ile Ile Leu Cys Leu Gly Val Phe Leu 1 5 10 15 acc gta tca ctctat ttg tac aat gga att gaa acc gga gct gaa gcg 96 Thr Val Ser Leu TyrLeu Tyr Asn Gly Ile Glu Thr Gly Ala Glu Ala 20 25 30 ctc acc aaa cga caagca aat gat tta cgg cgg aaa atc gga aat ttg 144 Leu Thr Lys Arg Gln AlaAsn Asp Leu Arg Arg Lys Ile Gly Asn Leu 35 40 45 gag cat gta gca gaa gaaaat gga aga acg ata gac cgc ttg gaa caa 192 Glu His Val Ala Glu Glu AsnGly Arg Thr Ile Asp Arg Leu Glu Gln 50 55 60 gaa gtt caa cga gca aaa gctgaa aaa tcg gta gat ttt gat gaa gaa 240 Glu Val Gln Arg Ala Lys Ala GluLys Ser Val Asp Phe Asp Glu Glu 65 70 75 80 aaa gaa aaa acg gaa gaa aaagaa gta gaa aaa gag gaa aaa gaa gtt 288 Lys Glu Lys Thr Glu Glu Lys GluVal Glu Lys Glu Glu Lys Glu Val 85 90 95 gca cca gtt cca gtt cga gga aatcgt ggt gaa atg gct cat att cat 336 Ala Pro Val Pro Val Arg Gly Asn ArgGly Glu Met Ala His Ile His 100 105 110 caa gta aag caa cat atc aag ccaact cca tcg atg aaa gat gtt tgt 384 Gln Val Lys Gln His Ile Lys Pro ThrPro Ser Met Lys Asp Val Cys 115 120 125 gga att aga gaa aac gtc agc attgct cat tca gac ctg cag atg ctc 432 Gly Ile Arg Glu Asn Val Ser Ile AlaHis Ser Asp Leu Gln Met Leu 130 135 140 gat ctc tat gac acc tgg aag ttcgaa aat cca gac gga ggt gta tgg 480 Asp Leu Tyr Asp Thr Trp Lys Phe GluAsn Pro Asp Gly Gly Val Trp 145 150 155 160 aaa caa gga tgg aaa att gaatac gat gca gag aaa gtc aaa tct ctt 528 Lys Gln Gly Trp Lys Ile Glu TyrAsp Ala Glu Lys Val Lys Ser Leu 165 170 175 cca cgt ttg gaa gtt att gtgata cct cat tct cat tgt gat ccc gga 576 Pro Arg Leu Glu Val Ile Val IlePro His Ser His Cys Asp Pro Gly 180 185 190 tgg att atg act ttc gaa gagtat tac aac aga caa act cgc aat att 624 Trp Ile Met Thr Phe Glu Glu TyrTyr Asn Arg Gln Thr Arg Asn Ile 195 200 205 ctt gat gga atg gct aaa catttg gca gaa aaa gac gaa atg cgg ttt 672 Leu Asp Gly Met Ala Lys His LeuAla Glu Lys Asp Glu Met Arg Phe 210 215 220 ata tat gca gaa ata tca tttttc gaa act tgg tgg aga gac cag gca 720 Ile Tyr Ala Glu Ile Ser Phe PheGlu Thr Trp Trp Arg Asp Gln Ala 225 230 235 240 gat gaa att aaa aag aaagtt aaa gga tat ttg gaa gca gga aag ttt 768 Asp Glu Ile Lys Lys Lys ValLys Gly Tyr Leu Glu Ala Gly Lys Phe 245 250 255 gaa att gtt act ggc ggatgg gtt atg aca gat gaa gct aat gca cat 816 Glu Ile Val Thr Gly Gly TrpVal Met Thr Asp Glu Ala Asn Ala His 260 265 270 tat cac tca atg atc actgaa ttg ttc gaa gga cat gaa tgg att caa 864 Tyr His Ser Met Ile Thr GluLeu Phe Glu Gly His Glu Trp Ile Gln 275 280 285 aat cat ttg gga aaa agcgcc att cca caa tct cat tgg tca att gat 912 Asn His Leu Gly Lys Ser AlaIle Pro Gln Ser His Trp Ser Ile Asp 290 295 300 cca ttc ggt tta tca ccatca atg cca cat ctt cta act tct gct aat 960 Pro Phe Gly Leu Ser Pro SerMet Pro His Leu Leu Thr Ser Ala Asn 305 310 315 320 ata acc aat gct gtaatt caa aga gtt cat tat tcg gtg aaa cgt gag 1008 Ile Thr Asn Ala Val IleGln Arg Val His Tyr Ser Val Lys Arg Glu 325 330 335 ctt gct ctg aaa aagaat ctt gaa ttc tac tgg aga caa tta ttt gga 1056 Leu Ala Leu Lys Lys AsnLeu Glu Phe Tyr Trp Arg Gln Leu Phe Gly 340 345 350 tca act gga cat cctgat ctt cgt tca cat att atg cct ttc tac tct 1104 Ser Thr Gly His Pro AspLeu Arg Ser His Ile Met Pro Phe Tyr Ser 355 360 365 tac gat ata cct catacg tgt ggc cca gaa ccg tct gtt tgc tgt caa 1152 Tyr Asp Ile Pro His ThrCys Gly Pro Glu Pro Ser Val Cys Cys Gln 370 375 380 ttc gat ttc cgt agaatg cca gaa ggt gga aaa tca tgt gat tgg gga 1200 Phe Asp Phe Arg Arg MetPro Glu Gly Gly Lys Ser Cys Asp Trp Gly 385 390 395 400 atc cct cca cagaaa att aac gat gac aat gtg gct cac aga gct gaa 1248 Ile Pro Pro Gln LysIle Asn Asp Asp Asn Val Ala His Arg Ala Glu 405 410 415 atg att tat gatcaa tat aga aag aaa agt caa ctt ttc aag aat aat 1296 Met Ile Tyr Asp GlnTyr Arg Lys Lys Ser Gln Leu Phe Lys Asn Asn 420 425 430 gtg att ttc caacca ctt gga gat gat ttc agg tac gac att gat ttt 1344 Val Ile Phe Gln ProLeu Gly Asp Asp Phe Arg Tyr Asp Ile Asp Phe 435 440 445 gaa tgg aat tcacaa tat gaa aac tat aag aaa ttg ttc gaa tac atg 1392 Glu Trp Asn Ser GlnTyr Glu Asn Tyr Lys Lys Leu Phe Glu Tyr Met 450 455 460 aat tcc aaa tcagaa tgg aat gtt cat gct caa ttc gga act ctt tct 1440 Asn Ser Lys Ser GluTrp Asn Val His Ala Gln Phe Gly Thr Leu Ser 465 470 475 480 gat tat ttcaag aag ctt gat act gca att tct gcg tct ggc gag caa 1488 Asp Tyr Phe LysLys Leu Asp Thr Ala Ile Ser Ala Ser Gly Glu Gln 485 490 495 ctt cca actttt tct gga gat ttc ttc act tat gcg gac aga gat gat 1536 Leu Pro Thr PheSer Gly Asp Phe Phe Thr Tyr Ala Asp Arg Asp Asp 500 505 510 cat tat tggagt gga tac ttc act tcc cgt cca ttc tat aaa cag ctt 1584 His Tyr Trp SerGly Tyr Phe Thr Ser Arg Pro Phe Tyr Lys Gln Leu 515 520 525 gat cgg gttctc caa cat tat tta aga tca gct gaa atc gcc ttt acc 1632 Asp Arg Val LeuGln His Tyr Leu Arg Ser Ala Glu Ile Ala Phe Thr 530 535 540 ctt gca aatatt gaa gaa gaa gga atg gtt gaa gcg aaa att ttt gag 1680 Leu Ala Asn IleGlu Glu Glu Gly Met Val Glu Ala Lys Ile Phe Glu 545 550 555 560 aag cttgtg act gct cga cga gct ctt tca ctt ttc caa cat cac gat 1728 Lys Leu ValThr Ala Arg Arg Ala Leu Ser Leu Phe Gln His His Asp 565 570 575 ggt gtaact ggt acg gca aaa gat cac gtc gtc ttg gat tat ggt cag 1776 Gly Val ThrGly Thr Ala Lys Asp His Val Val Leu Asp Tyr Gly Gln 580 585 590 aaa atgatt gat gct ttg aac gca tgt gag gat att ctt tcg gaa gct 1824 Lys Met IleAsp Ala Leu Asn Ala Cys Glu Asp Ile Leu Ser Glu Ala 595 600 605 ctt gttgta ttg ctg gga att gat tca acg aat aag atg cag atg gat 1872 Leu Val ValLeu Leu Gly Ile Asp Ser Thr Asn Lys Met Gln Met Asp 610 615 620 gag cataga gtt aat gaa aac ctt cta ccc gaa aaa cgt gtc tat aaa 1920 Glu His ArgVal Asn Glu Asn Leu Leu Pro Glu Lys Arg Val Tyr Lys 625 630 635 640 attggg caa aac gtc gta ttg ttc aat act tta tct aga aat cgc aac 1968 Ile GlyGln Asn Val Val Leu Phe Asn Thr Leu Ser Arg Asn Arg Asn 645 650 655 gagcca att tgt att caa gtt gat tct ctt gac gct ggt gtc gaa gct 2016 Glu ProIle Cys Ile Gln Val Asp Ser Leu Asp Ala Gly Val Glu Ala 660 665 670 gatcct cca att aaa aaa caa caa gtt tcg ccg gtt att gca tat gat 2064 Asp ProPro Ile Lys Lys Gln Gln Val Ser Pro Val Ile Ala Tyr Asp 675 680 685 gaagag aag aaa acg ctt gtt gtc aaa aac gga ata ttc gaa ctt tgc 2112 Glu GluLys Lys Thr Leu Val Val Lys Asn Gly Ile Phe Glu Leu Cys 690 695 700 ttcatg tta tca ctt gga cca atg gag tct gtc agt ttc aga ctt gtg 2160 Phe MetLeu Ser Leu Gly Pro Met Glu Ser Val Ser Phe Arg Leu Val 705 710 715 720aaa aat aca aca aca tcc aaa gtt gaa ata atc acc aat aat gcg gca 2208 LysAsn Thr Thr Thr Ser Lys Val Glu Ile Ile Thr Asn Asn Ala Ala 725 730 735gaa ttc aaa gaa aca agt ttt aaa tct tca tcc act tct gga gac ttt 2256 GluPhe Lys Glu Thr Ser Phe Lys Ser Ser Ser Thr Ser Gly Asp Phe 740 745 750act gtg aaa aac gac aaa gtt gaa gct gaa ttt gat gga gaa aat gga 2304 ThrVal Lys Asn Asp Lys Val Glu Ala Glu Phe Asp Gly Glu Asn Gly 755 760 765atg att aaa aga gct acc agt ctt gtt gat gat aaa cca att gat ttg 2352 MetIle Lys Arg Ala Thr Ser Leu Val Asp Asp Lys Pro Ile Asp Leu 770 775 780aat tct cac ttt att cat tat gga gca cgg aag tca aag aga aag ttc 2400 AsnSer His Phe Ile His Tyr Gly Ala Arg Lys Ser Lys Arg Lys Phe 785 790 795800 gca aat gga aat gaa gac aac ccg gct ggc gca tac ctg ttc ctt ccc 2448Ala Asn Gly Asn Glu Asp Asn Pro Ala Gly Ala Tyr Leu Phe Leu Pro 805 810815 gat gga gaa gct aga gaa ctc aaa aaa caa tca agt gat tgg ata ttg 2496Asp Gly Glu Ala Arg Glu Leu Lys Lys Gln Ser Ser Asp Trp Ile Leu 820 825830 gta aaa gga gaa gtt gtt caa aaa gtg ttt gca act cca aac aat gat 2544Val Lys Gly Glu Val Val Gln Lys Val Phe Ala Thr Pro Asn Asn Asp 835 840845 ctg aaa ata ttg caa acg tac aca ctt tat caa ggg ctt cca tgg att 2592Leu Lys Ile Leu Gln Thr Tyr Thr Leu Tyr Gln Gly Leu Pro Trp Ile 850 855860 gat ttg gat aat gaa gtt gat gta cgt tcc aag gag aat ttc gag ttg 2640Asp Leu Asp Asn Glu Val Asp Val Arg Ser Lys Glu Asn Phe Glu Leu 865 870875 880 gca ctg aga ttc agt tct tca gta aat agt ggt gat gag ttt ttc act2688 Ala Leu Arg Phe Ser Ser Ser Val Asn Ser Gly Asp Glu Phe Phe Thr 885890 895 gat ctc aat gga atg caa atg ata aaa agg aga cga caa act aaa tta2736 Asp Leu Asn Gly Met Gln Met Ile Lys Arg Arg Arg Gln Thr Lys Leu 900905 910 cca aca cag gcc aat ttc tat ccc atg tct gct ggt gtt tac att gaa2784 Pro Thr Gln Ala Asn Phe Tyr Pro Met Ser Ala Gly Val Tyr Ile Glu 915920 925 gac gat act acc aga atg tca att cat tcg gca cag gct ctc gga gtt2832 Asp Asp Thr Thr Arg Met Ser Ile His Ser Ala Gln Ala Leu Gly Val 930935 940 agc agt ctc tcg tcg gga caa att gaa ata atg ctt gat cga cga ctt2880 Ser Ser Leu Ser Ser Gly Gln Ile Glu Ile Met Leu Asp Arg Arg Leu 945950 955 960 agt tca gat gac aac aga ggt ctt cag caa gga gtt aga gac aacaaa 2928 Ser Ser Asp Asp Asn Arg Gly Leu Gln Gln Gly Val Arg Asp Asn Lys965 970 975 cga aca gtt gca cat ttc cgt att gtt att gag ccg atg tct tcatcg 2976 Arg Thr Val Ala His Phe Arg Ile Val Ile Glu Pro Met Ser Ser Ser980 985 990 agt ggt aat aag aag gaa gaa cga gtt gga ttc cat tca cat gttggt 3024 Ser Gly Asn Lys Lys Glu Glu Arg Val Gly Phe His Ser His Val Gly995 1000 1005 cat ctc gct acg tgg tct ctt cat tat cct ctt gtc aaa atgatt gga 3072 His Leu Ala Thr Trp Ser Leu His Tyr Pro Leu Val Lys Met IleGly 1010 1015 1020 gat gca aca cca aaa tct att tct tcg aaa aat gtg gaacaa gag ctg 3120 Asp Ala Thr Pro Lys Ser Ile Ser Ser Lys Asn Val Glu GlnGlu Leu 1025 1030 1035 1040 aac tgt gac ctg cat cta gtg aca ttt aga acactg gca tcg ccg aca 3168 Asn Cys Asp Leu His Leu Val Thr Phe Arg Thr LeuAla Ser Pro Thr 1045 1050 1055 act tac gaa gcc aac gaa aga tct acg gcagct gag aag aaa gca gcg 3216 Thr Tyr Glu Ala Asn Glu Arg Ser Thr Ala AlaGlu Lys Lys Ala Ala 1060 1065 1070 atg gtg atg cat aga gtt gtt cca gactgt aga tcc agg ctt acc ctc 3264 Met Val Met His Arg Val Val Pro Asp CysArg Ser Arg Leu Thr Leu 1075 1080 1085 cca gac acg tca tgc tta gct actgga tta gaa att gag cca ctc aaa 3312 Pro Asp Thr Ser Cys Leu Ala Thr GlyLeu Glu Ile Glu Pro Leu Lys 1090 1095 1100 ttg atc tcg aca ctg aag tctgcg aaa aaa acg tca cta acc aat ctt 3360 Leu Ile Ser Thr Leu Lys Ser AlaLys Lys Thr Ser Leu Thr Asn Leu 1105 1110 1115 1120 tat gaa gga aac aaggct gaa caa ttc cga ctc caa cca aac gat att 3408 Tyr Glu Gly Asn Lys AlaGlu Gln Phe Arg Leu Gln Pro Asn Asp Ile 1125 1130 1135 tcc agt att cttgta tca ttt taa 3432 Ser Ser Ile Leu Val Ser Phe 1140 51 3450 DNA Cionaintestinalis CDS (1)..(3447) 51 atg aag ctc aaa cgc cag ttc tta ttc tttggt gga att ctg ttc ttc 48 Met Lys Leu Lys Arg Gln Phe Leu Phe Phe GlyGly Ile Leu Phe Phe 1 5 10 15 ggg agt atc tgg ttt atg ata ggt caa cttgac act cct aat tcg cca 96 Gly Ser Ile Trp Phe Met Ile Gly Gln Leu AspThr Pro Asn Ser Pro 20 25 30 cag aaa gtc aaa ttc tcg gaa ggc agt gaa aatgac caa gtt cga acc 144 Gln Lys Val Lys Phe Ser Glu Gly Ser Glu Asn AspGln Val Arg Thr 35 40 45 ctt caa gac aaa ctt agt ctg gtg gaa aaa gaa ttgtta gaa aat cgt 192 Leu Gln Asp Lys Leu Ser Leu Val Glu Lys Glu Leu LeuGlu Asn Arg 50 55 60 aaa ata atg cac aag gtg aaa gat agt cta cag gat atgaca ccc atg 240 Lys Ile Met His Lys Val Lys Asp Ser Leu Gln Asp Met ThrPro Met 65 70 75 80 aaa aat gtt cat gtg cct atg cag cgc gga gaa ata agaaac aac gtc 288 Lys Asn Val His Val Pro Met Gln Arg Gly Glu Ile Arg AsnAsn Val 85 90 95 aat aaa cct gtg cta cca ctt ata atg ccc aag caa ttt gcgaat gac 336 Asn Lys Pro Val Leu Pro Leu Ile Met Pro Lys Gln Phe Ala AsnAsp 100 105 110 tcc cga atg agt gac acg tgt cct gtg ctc tcg tac tcc ggtggc aag 384 Ser Arg Met Ser Asp Thr Cys Pro Val Leu Ser Tyr Ser Gly GlyLys 115 120 125 tcc gat gtt aac atg att aac gtg tat gat cat ctt cca tttgat gat 432 Ser Asp Val Asn Met Ile Asn Val Tyr Asp His Leu Pro Phe AspAsp 130 135 140 cca gat ggt gga gtt tgg aaa caa ggt tgg gac atc cag acatcg gat 480 Pro Asp Gly Gly Val Trp Lys Gln Gly Trp Asp Ile Gln Thr SerAsp 145 150 155 160 cag gaa tgg gct ggg aga aaa ttg aaa gtg ttc att gtccct cac tca 528 Gln Glu Trp Ala Gly Arg Lys Leu Lys Val Phe Ile Val ProHis Ser 165 170 175 cat aat gat cct ggt tgg tta aag acg gtg gaa aga tacttc agc gat 576 His Asn Asp Pro Gly Trp Leu Lys Thr Val Glu Arg Tyr PheSer Asp 180 185 190 caa aca caa cat att ctc aat aat att gtg gat gct ttgagt caa gac 624 Gln Thr Gln His Ile Leu Asn Asn Ile Val Asp Ala Leu SerGln Asp 195 200 205 cct gca agg aag ttt atc tgg gca gag atg tcg tat ctctca atg tgg 672 Pro Ala Arg Lys Phe Ile Trp Ala Glu Met Ser Tyr Leu SerMet Trp 210 215 220 tgg gac att gcc aca cct gat cgt aag cag aaa atg cagaca ctc gtg 720 Trp Asp Ile Ala Thr Pro Asp Arg Lys Gln Lys Met Gln ThrLeu Val 225 230 235 240 aag aat gga cag ctt gag ata gtt acg ggt ggt tgggtc atg aat gat 768 Lys Asn Gly Gln Leu Glu Ile Val Thr Gly Gly Trp ValMet Asn Asp 245 250 255 gaa gca aac act cat tac ttt gct atg att gat caactc att gaa ggt 816 Glu Ala Asn Thr His Tyr Phe Ala Met Ile Asp Gln LeuIle Glu Gly 260 265 270 atg gaa tgg ttg agg cga aca ttg aat gtt gtt ccaaaa agt ggg tgg 864 Met Glu Trp Leu Arg Arg Thr Leu Asn Val Val Pro LysSer Gly Trp 275 280 285 gcg att gat ccc ttt ggt cac acc ccc acc atg gcttat ata ctg aaa 912 Ala Ile Asp Pro Phe Gly His Thr Pro Thr Met Ala TyrIle Leu Lys 290 295 300 cag atg aag ttc aaa aac atg ctg ata caa aga gtccat tat gca gtg 960 Gln Met Lys Phe Lys Asn Met Leu Ile Gln Arg Val HisTyr Ala Val 305 310 315 320 aag aag tat ctt gct cag gaa aag tct ctg gaattc aga tgg aga caa 1008 Lys Lys Tyr Leu Ala Gln Glu Lys Ser Leu Glu PheArg Trp Arg Gln 325 330 335 atg tgg gat tca gct tca agt aca gac atg atgtgt cat ctc atg cct 1056 Met Trp Asp Ser Ala Ser Ser Thr Asp Met Met CysHis Leu Met Pro 340 345 350 ttc tat tca tat gat gtt cct cat act tgt ggccca gac ccc aag att 1104 Phe Tyr Ser Tyr Asp Val Pro His Thr Cys Gly ProAsp Pro Lys Ile 355 360 365 tgc tgc cag ttt gat ttt gct cgc tta ccc ggcggc aag ata acc tgc 1152 Cys Cys Gln Phe Asp Phe Ala Arg Leu Pro Gly GlyLys Ile Thr Cys 370 375 380 cca tgg aaa gtt cct cct gtt gcc atc act gactcc aat gta gaa aca 1200 Pro Trp Lys Val Pro Pro Val Ala Ile Thr Asp SerAsn Val Glu Thr 385 390 395 400 cga gcc gga ata cta ctt gac caa tat agaaaa aag tca aaa ctc ttc 1248 Arg Ala Gly Ile Leu Leu Asp Gln Tyr Arg LysLys Ser Lys Leu Phe 405 410 415 aaa agt gac acc ctg ctt att ata tta ggagat gat ttt cgt tat tcg 1296 Lys Ser Asp Thr Leu Leu Ile Ile Leu Gly AspAsp Phe Arg Tyr Ser 420 425 430 ctg agc aag gaa acc aac gat cag ttt gacaac tac gct cga att atc 1344 Leu Ser Lys Glu Thr Asn Asp Gln Phe Asp AsnTyr Ala Arg Ile Ile 435 440 445 tcg tat gtg aat tcg cac cca gag tta aacgca aaa ctt cag ttt gga 1392 Ser Tyr Val Asn Ser His Pro Glu Leu Asn AlaLys Leu Gln Phe Gly 450 455 460 aca tta tcc gaa tat ttt gat gcc atg aaatct gaa gtg ggg gga gag 1440 Thr Leu Ser Glu Tyr Phe Asp Ala Met Lys SerGlu Val Gly Gly Glu 465 470 475 480 gaa aaa ctc cca gct tta agt ggt gatttc ttc act tat gct gat aga 1488 Glu Lys Leu Pro Ala Leu Ser Gly Asp PhePhe Thr Tyr Ala Asp Arg 485 490 495 gaa gat cac tat tgg agt ggt tac tacact tca cgg cct tac cac aaa 1536 Glu Asp His Tyr Trp Ser Gly Tyr Tyr ThrSer Arg Pro Tyr His Lys 500 505 510 atg cag gag aga gtc ctg gaa agc cacctt cga gga gca gaa atg ttg 1584 Met Gln Glu Arg Val Leu Glu Ser His LeuArg Gly Ala Glu Met Leu 515 520 525 ttt gcg ctc tca tgg ccc aaa atc cagtgg aca gga ctt ggt gaa aca 1632 Phe Ala Leu Ser Trp Pro Lys Ile Gln TrpThr Gly Leu Gly Glu Thr 530 535 540 ttt tca cat gaa ctt tac cca ctg ctggtc caa gca cgt caa aat ctt 1680 Phe Ser His Glu Leu Tyr Pro Leu Leu ValGln Ala Arg Gln Asn Leu 545 550 555 560 ggt ttg ttt caa cac cac gat ggtata aca ggc aca gca aag gat cat 1728 Gly Leu Phe Gln His His Asp Gly IleThr Gly Thr Ala Lys Asp His 565 570 575 gtt gtt gtt gat tac ggg aat aaactc atg aag agt gtt atg gat gca 1776 Val Val Val Asp Tyr Gly Asn Lys LeuMet Lys Ser Val Met Asp Ala 580 585 590 aag aag gta att tca tac agt gcccaa gtt ctg ttg caa gaa atg atc 1824 Lys Lys Val Ile Ser Tyr Ser Ala GlnVal Leu Leu Gln Glu Met Ile 595 600 605 acg ttt gat cca aat acc atg gtactt aac tat gat gag gtg tat caa 1872 Thr Phe Asp Pro Asn Thr Met Val LeuAsn Tyr Asp Glu Val Tyr Gln 610 615 620 gct cag aac caa caa cct gcg cctgtg gtt gtt aag cta cca acg aag 1920 Ala Gln Asn Gln Gln Pro Ala Pro ValVal Val Lys Leu Pro Thr Lys 625 630 635 640 aat gaa gaa gcg cgg aaa gtcgtt ctc tac aac tct ctg gat tac gac 1968 Asn Glu Glu Ala Arg Lys Val ValLeu Tyr Asn Ser Leu Asp Tyr Asp 645 650 655 aga act ggt gtc gtg cgt ctaatt gtt acg tca ccc gac gtg gtt gtg 2016 Arg Thr Gly Val Val Arg Leu IleVal Thr Ser Pro Asp Val Val Val 660 665 670 atg tca gaa aac aaa aac gtcgtt cca tcg caa acc agt ccg atc tgg 2064 Met Ser Glu Asn Lys Asn Val ValPro Ser Gln Thr Ser Pro Ile Trp 675 680 685 tca gat tcg acg gag atc cgcaca gac cag ttt gaa ctg gtt ttc ctt 2112 Ser Asp Ser Thr Glu Ile Arg ThrAsp Gln Phe Glu Leu Val Phe Leu 690 695 700 tca act gtt ccc gcg ata ggactg gcg gtg tac aag ata tgg gaa gac 2160 Ser Thr Val Pro Ala Ile Gly LeuAla Val Tyr Lys Ile Trp Glu Asp 705 710 715 720 aac gac gta gca gac accacg cac tca act gtt aag ttt atc aac ccg 2208 Asn Asp Val Ala Asp Thr ThrHis Ser Thr Val Lys Phe Ile Asn Pro 725 730 735 aga gtt ggg ttt tcg aaacga acc cgc agt aag ttt gta ctc gac gtt 2256 Arg Val Gly Phe Ser Lys ArgThr Arg Ser Lys Phe Val Leu Asp Val 740 745 750 gag gat agc ggg gag tttacc atc atg aat gac caa tta gtt gcg cat 2304 Glu Asp Ser Gly Glu Phe ThrIle Met Asn Asp Gln Leu Val Ala His 755 760 765 ttc tct gga caa aac gggatg ctg cag tca gtc acc act gtg cgt gac 2352 Phe Ser Gly Gln Asn Gly MetLeu Gln Ser Val Thr Thr Val Arg Asp 770 775 780 aac gtt aaa acg cag ctcgga att gaa ttc gtc gct tat act tct cgt 2400 Asn Val Lys Thr Gln Leu GlyIle Glu Phe Val Ala Tyr Thr Ser Arg 785 790 795 800 aat aag aaa gac aagagc ggc gct tac ttg ttc ctg cct gct gga cca 2448 Asn Lys Lys Asp Lys SerGly Ala Tyr Leu Phe Leu Pro Ala Gly Pro 805 810 815 gca caa ccg cat gtaaca gaa tcc cac cga ccg tta gta agg atc atc 2496 Ala Gln Pro His Val ThrGlu Ser His Arg Pro Leu Val Arg Ile Ile 820 825 830 agg ggt cca gtg atgtca acg gtg cat gtt cta cta ccg aac gtt ctg 2544 Arg Gly Pro Val Met SerThr Val His Val Leu Leu Pro Asn Val Leu 835 840 845 cat aaa gtt acc ctatac acc ggt act ggt gca ggc acg cag tct tta 2592 His Lys Val Thr Leu TyrThr Gly Thr Gly Ala Gly Thr Gln Ser Leu 850 855 860 ggc gtc cac gtc tctaac gac gtc gac gtt aga act ggc tac gac aac 2640 Gly Val His Val Ser AsnAsp Val Asp Val Arg Thr Gly Tyr Asp Asn 865 870 875 880 aaa gaa ctc agtatg agg tta aac agc gaa gtt tta tcg gga agc aaa 2688 Lys Glu Leu Ser MetArg Leu Asn Ser Glu Val Leu Ser Gly Ser Lys 885 890 895 ttc ttt acg gattta aac ggt ttc caa att caa ccc cga acc acg tat 2736 Phe Phe Thr Asp LeuAsn Gly Phe Gln Ile Gln Pro Arg Thr Thr Tyr 900 905 910 tct aaa ctg ccacta caa gca aac ttc tac cca ata ccc aca atg gcg 2784 Ser Lys Leu Pro LeuGln Ala Asn Phe Tyr Pro Ile Pro Thr Met Ala 915 920 925 ttc ata caa gacgaa aaa tca aga tta act ttg atg acg gcc caa cca 2832 Phe Ile Gln Asp GluLys Ser Arg Leu Thr Leu Met Thr Ala Gln Pro 930 935 940 ctg ggt gtt gcctca ctg aag tca ggt caa ctt gag gtg gtt ttg gat 2880 Leu Gly Val Ala SerLeu Lys Ser Gly Gln Leu Glu Val Val Leu Asp 945 950 955 960 cgc cgt ttaatg cag gac gac aac agg ggg gtg ggt caa ggt gtg aaa 2928 Arg Arg Leu MetGln Asp Asp Asn Arg Gly Val Gly Gln Gly Val Lys 965 970 975 gat aat ttacca act cct gag agt ttc gtg atc atg ctg gaa aga tgg 2976 Asp Asn Leu ProThr Pro Glu Ser Phe Val Ile Met Leu Glu Arg Trp 980 985 990 acc gct attgca gcg aaa gaa agc aaa tcg tca gcg aag ctc gcg tat 3024 Thr Ala Ile AlaAla Lys Glu Ser Lys Ser Ser Ala Lys Leu Ala Tyr 995 1000 1005 cca tctatg gct gtg tat cag tca tca tgg gaa ttg cta cac cca ata 3072 Pro Ser MetAla Val Tyr Gln Ser Ser Trp Glu Leu Leu His Pro Ile 1010 1015 1020 cgtcca atg tcg gta aat ggg ccg gta cat ttg aaa gaa gat tac cgc 3120 Arg ProMet Ser Val Asn Gly Pro Val His Leu Lys Glu Asp Tyr Arg 1025 1030 10351040 tcg ctg cca cag cct tta cca tgc gac gtg cac gtg tta aac ttg cga3168 Ser Leu Pro Gln Pro Leu Pro Cys Asp Val His Val Leu Asn Leu Arg1045 1050 1055 gca att cat tct aaa gat gca gtt gcc cct acc gac caa tcggct ctg 3216 Ala Ile His Ser Lys Asp Ala Val Ala Pro Thr Asp Gln Ser AlaLeu 1060 1065 1070 ctt cta cac aca gtt ggg cgc gaa tgc tcc ttg gac gcggat aag tat 3264 Leu Leu His Thr Val Gly Arg Glu Cys Ser Leu Asp Ala AspLys Tyr 1075 1080 1085 ttc cac cca acg tgc ctc atg cac ggc gtc gag aaattg gct atc acg 3312 Phe His Pro Thr Cys Leu Met His Gly Val Glu Lys LeuAla Ile Thr 1090 1095 1100 atc tcg acg ctt ttt act aac tct ggc atg cggaag acg tcg ctg tcc 3360 Ile Ser Thr Leu Phe Thr Asn Ser Gly Met Arg LysThr Ser Leu Ser 1105 1110 1115 1120 tta caa cac gac ggc tcg ttg ctg gacaac caa ggc ggt att aca gtt 3408 Leu Gln His Asp Gly Ser Leu Leu Asp AsnGln Gly Gly Ile Thr Val 1125 1130 1135 tcc cca atg gag ata caa gct tacaaa ata gta ctg acg taa 3450 Ser Pro Met Glu Ile Gln Ala Tyr Lys Ile ValLeu Thr 1140 1145 52 3327 DNA Drosophila sp. CDS (1)..(3324) 52 atg ttgcga ata cgt cgg cgg ttc gct ttg gta att tgc tcc ggc tgc 48 Met Leu ArgIle Arg Arg Arg Phe Ala Leu Val Ile Cys Ser Gly Cys 1 5 10 15 ctg ctggtt ttc ctc agc ctg tac ata atc ctc aat ttt gcg gcg ccg 96 Leu Leu ValPhe Leu Ser Leu Tyr Ile Ile Leu Asn Phe Ala Ala Pro 20 25 30 gca gcc acccag ata aag ccc aac tat gag aac att gag aac aag ctg 144 Ala Ala Thr GlnIle Lys Pro Asn Tyr Glu Asn Ile Glu Asn Lys Leu 35 40 45 cat gag ctg gaaaat ggt ttg cag gag cac ggg gag gag atg cgg aat 192 His Glu Leu Glu AsnGly Leu Gln Glu His Gly Glu Glu Met Arg Asn 50 55 60 ctc agg gcg cgt ctggcc aaa aca tcc aat cgc gac gat cca ata aga 240 Leu Arg Ala Arg Leu AlaLys Thr Ser Asn Arg Asp Asp Pro Ile Arg 65 70 75 80 cct cca ctt aaa gtggct cgt tcc ccg agg cca ggg caa tgc caa gat 288 Pro Pro Leu Lys Val AlaArg Ser Pro Arg Pro Gly Gln Cys Gln Asp 85 90 95 gtg gtc caa gac gtg cccaat gtg gat gta cag atg ctg gag cta tac 336 Val Val Gln Asp Val Pro AsnVal Asp Val Gln Met Leu Glu Leu Tyr 100 105 110 gat cgc atg tcc ttc aaggac ata gat gga ggc gtg tgg aaa cag ggc 384 Asp Arg Met Ser Phe Lys AspIle Asp Gly Gly Val Trp Lys Gln Gly 115 120 125 tgg aac att aag tac gatcca ctg aag tac aac gcc cat cac aaa cta 432 Trp Asn Ile Lys Tyr Asp ProLeu Lys Tyr Asn Ala His His Lys Leu 130 135 140 aaa gtc ttc gtt gtg ccgcac tcg cac aac gat cct gga tgg att cag 480 Lys Val Phe Val Val Pro HisSer His Asn Asp Pro Gly Trp Ile Gln 145 150 155 160 acg ttt gag gaa tactac cag cac gac acc aag cac atc ctg tcc aat 528 Thr Phe Glu Glu Tyr TyrGln His Asp Thr Lys His Ile Leu Ser Asn 165 170 175 gca cta cgg cat ctgcac gac aat ccc gag atg aag ttc atc tgg gcg 576 Ala Leu Arg His Leu HisAsp Asn Pro Glu Met Lys Phe Ile Trp Ala 180 185 190 gaa atc tcc tac tttgct cgg ttc tat cac gat ttg gga gag aac aaa 624 Glu Ile Ser Tyr Phe AlaArg Phe Tyr His Asp Leu Gly Glu Asn Lys 195 200 205 aag ctg cag atg aagtcc att gta aag aat gga cag ttg gaa ttt gtg 672 Lys Leu Gln Met Lys SerIle Val Lys Asn Gly Gln Leu Glu Phe Val 210 215 220 act gga gga tgg gtaatg ccg gac gag gcc aac tcc cac tgg cga aac 720 Thr Gly Gly Trp Val MetPro Asp Glu Ala Asn Ser His Trp Arg Asn 225 230 235 240 gta ctg ctg cagctg acc gaa ggg caa aca tgg ttg aag caa ttc atg 768 Val Leu Leu Gln LeuThr Glu Gly Gln Thr Trp Leu Lys Gln Phe Met 245 250 255 aat gtc aca cccact gct tcc tgg gcc atc gat ccc ttc gga cac agt 816 Asn Val Thr Pro ThrAla Ser Trp Ala Ile Asp Pro Phe Gly His Ser 260 265 270 ccc act atg ccgtac att ttg cag aag agt ggt ttc aag aat atg ctt 864 Pro Thr Met Pro TyrIle Leu Gln Lys Ser Gly Phe Lys Asn Met Leu 275 280 285 atc caa agg acgcac tat tcg gtt aag aag gaa ctg gcc caa cag cga 912 Ile Gln Arg Thr HisTyr Ser Val Lys Lys Glu Leu Ala Gln Gln Arg 290 295 300 cag ctt gag ttcctg tgg cgc cag atc tgg gac aac aaa ggg gac aca 960 Gln Leu Glu Phe LeuTrp Arg Gln Ile Trp Asp Asn Lys Gly Asp Thr 305 310 315 320 gct ctc ttcacc cac atg atg ccc ttc tac tcg tac gac att cct cat 1008 Ala Leu Phe ThrHis Met Met Pro Phe Tyr Ser Tyr Asp Ile Pro His 325 330 335 acc tgt ggtcca gat ccc aag gtt tgc tgt cag ttc gat ttc aaa cga 1056 Thr Cys Gly ProAsp Pro Lys Val Cys Cys Gln Phe Asp Phe Lys Arg 340 345 350 atg ggc tccttc ggt ttg agt tgt cca tgg aag gtg ccg ccg cgt aca 1104 Met Gly Ser PheGly Leu Ser Cys Pro Trp Lys Val Pro Pro Arg Thr 355 360 365 atc agt gatcaa aat gtg gca gca cgc tca gat ctg ctg gtt gat cag 1152 Ile Ser Asp GlnAsn Val Ala Ala Arg Ser Asp Leu Leu Val Asp Gln 370 375 380 tgg aag aagaag gcc gag ctg tat cgc aca aac gtg ctg ctg att ccg 1200 Trp Lys Lys LysAla Glu Leu Tyr Arg Thr Asn Val Leu Leu Ile Pro 385 390 395 400 ttg ggtgac gac ttc cgc ttc aag cag aac acc gag tgg gat gtg cag 1248 Leu Gly AspAsp Phe Arg Phe Lys Gln Asn Thr Glu Trp Asp Val Gln 405 410 415 cgc gtgaac tac gaa agg ctg ttc gaa cac atc aac agc cag gcc cac 1296 Arg Val AsnTyr Glu Arg Leu Phe Glu His Ile Asn Ser Gln Ala His 420 425 430 ttc aatgtc cag gcg cag ttc ggc aca ctg cag gaa tac ttt gat gca 1344 Phe Asn ValGln Ala Gln Phe Gly Thr Leu Gln Glu Tyr Phe Asp Ala 435 440 445 gtg caccag gcg gaa agg gcg gga caa gcc gag ttt ccc acg cta agc 1392 Val His GlnAla Glu Arg Ala Gly Gln Ala Glu Phe Pro Thr Leu Ser 450 455 460 ggt gacttt ttc aca tac gcc gat cga tcg gat aac tat tgg agt ggc 1440 Gly Asp PhePhe Thr Tyr Ala Asp Arg Ser Asp Asn Tyr Trp Ser Gly 465 470 475 480 tactac aca tcc cgc ccg tat cat aag cgc atg gac cgc gtc ctg atg 1488 Tyr TyrThr Ser Arg Pro Tyr His Lys Arg Met Asp Arg Val Leu Met 485 490 495 cactat gta cgt gca gca gaa atg ctt tcc gcc tgg cac tcc tgg gac 1536 His TyrVal Arg Ala Ala Glu Met Leu Ser Ala Trp His Ser Trp Asp 500 505 510 ggtatg gcc cgc atc gag gaa cgt ctg gag cag gcc cgc agg gag ctg 1584 Gly MetAla Arg Ile Glu Glu Arg Leu Glu Gln Ala Arg Arg Glu Leu 515 520 525 tcattg ttc cag cac cac gac ggt ata act ggc aca gca aaa acg cac 1632 Ser LeuPhe Gln His His Asp Gly Ile Thr Gly Thr Ala Lys Thr His 530 535 540 gtagtc gtc gac tac gag caa cgc atg cag gaa gct tta aaa gcc tgt 1680 Val ValVal Asp Tyr Glu Gln Arg Met Gln Glu Ala Leu Lys Ala Cys 545 550 555 560caa atg gta atg caa cag tcg gtc tac cga ttg ctg aca aag ccc tcc 1728 GlnMet Val Met Gln Gln Ser Val Tyr Arg Leu Leu Thr Lys Pro Ser 565 570 575atc tac agt ccg gac ttc agt ttc tcg tac ttt acg ctc gac gac tcc 1776 IleTyr Ser Pro Asp Phe Ser Phe Ser Tyr Phe Thr Leu Asp Asp Ser 580 585 590cgc tgg cca gga tct ggt gtg gag gac agt cga acc acc ata ata ctg 1824 ArgTrp Pro Gly Ser Gly Val Glu Asp Ser Arg Thr Thr Ile Ile Leu 595 600 605ggc gag gat ata ctg ccc tcc aag cat gtg gtg atg cac aac acc ctg 1872 GlyGlu Asp Ile Leu Pro Ser Lys His Val Val Met His Asn Thr Leu 610 615 620ccc cac tgg cgg gag cag ctg gtg gac ttt tat gta tcc agt ccg ttt 1920 ProHis Trp Arg Glu Gln Leu Val Asp Phe Tyr Val Ser Ser Pro Phe 625 630 635640 gta agc gtt acc gac ttg gca aac aat ccg gtg gag gct cag gtg tcc 1968Val Ser Val Thr Asp Leu Ala Asn Asn Pro Val Glu Ala Gln Val Ser 645 650655 ccg gtg tgg agc tgg cac cac gac aca ctc aca aag act atc cac cca 2016Pro Val Trp Ser Trp His His Asp Thr Leu Thr Lys Thr Ile His Pro 660 665670 caa ggc tcc acc acc aag tac cgc atc atc ttc aag gct cgg gtg ccg 2064Gln Gly Ser Thr Thr Lys Tyr Arg Ile Ile Phe Lys Ala Arg Val Pro 675 680685 ccc atg ggc ttg gcc acc tac gtt tta acc atc tcc gat tcc aag cca 2112Pro Met Gly Leu Ala Thr Tyr Val Leu Thr Ile Ser Asp Ser Lys Pro 690 695700 gag cac acc tcg tat gca tcg aat ctc ttg ctc cgt aaa aac ccg act 2160Glu His Thr Ser Tyr Ala Ser Asn Leu Leu Leu Arg Lys Asn Pro Thr 705 710715 720 tcg tta cca ttg ggc caa tat ccg gag gat gtg aag ttt ggc gat cct2208 Ser Leu Pro Leu Gly Gln Tyr Pro Glu Asp Val Lys Phe Gly Asp Pro 725730 735 cga gag atc tca ttg cgg gtt ggt aac gga ccc acc ttg gcc ttt tcg2256 Arg Glu Ile Ser Leu Arg Val Gly Asn Gly Pro Thr Leu Ala Phe Ser 740745 750 gag cag ggt ctc ctt aag tcc att cag ctt act cag gat agc cca cat2304 Glu Gln Gly Leu Leu Lys Ser Ile Gln Leu Thr Gln Asp Ser Pro His 755760 765 gta ccg gtg cac ttc aag ttc ctc aag tat ggc gtt cga tcg cat ggc2352 Val Pro Val His Phe Lys Phe Leu Lys Tyr Gly Val Arg Ser His Gly 770775 780 gat aga tcc ggt gcc tat ctg ttc ctg ccc aat gga cca gct tcg cca2400 Asp Arg Ser Gly Ala Tyr Leu Phe Leu Pro Asn Gly Pro Ala Ser Pro 785790 795 800 gtc gag ctt ggc cag cca gtg gtc ctg gtg act aag ggc aaa ctggag 2448 Val Glu Leu Gly Gln Pro Val Val Leu Val Thr Lys Gly Lys Leu Glu805 810 815 tcg tcc gtg agc gtg gga ctt ccg agc gtg gtg cac cag acg ataatg 2496 Ser Ser Val Ser Val Gly Leu Pro Ser Val Val His Gln Thr Ile Met820 825 830 cgc ggt ggt gca cct gag att cgc aat ctg gtg gat ata ggc tcactg 2544 Arg Gly Gly Ala Pro Glu Ile Arg Asn Leu Val Asp Ile Gly Ser Leu835 840 845 gac aac acg gag atc gtg atg cgc ttg gag acg cat atc gac agcggc 2592 Asp Asn Thr Glu Ile Val Met Arg Leu Glu Thr His Ile Asp Ser Gly850 855 860 gat atc ttc tac acg gat ctc aat gga ttg caa ttt atc aag aggcgg 2640 Asp Ile Phe Tyr Thr Asp Leu Asn Gly Leu Gln Phe Ile Lys Arg Arg865 870 875 880 cgt ttg gac aaa tta cct ttg cag gcc aac tat tat ccc atacct tct 2688 Arg Leu Asp Lys Leu Pro Leu Gln Ala Asn Tyr Tyr Pro Ile ProSer 885 890 895 ggt atg ttc att gag gat gcc aat acg cga ctc act ctc ctcacg ggt 2736 Gly Met Phe Ile Glu Asp Ala Asn Thr Arg Leu Thr Leu Leu ThrGly 900 905 910 caa ccg ctg ggt gga tct tct ctg gcc tcg ggc gag cta gagatt atg 2784 Gln Pro Leu Gly Gly Ser Ser Leu Ala Ser Gly Glu Leu Glu IleMet 915 920 925 caa gat cgt cgc ctg gcc agc gat gat gaa cgc ggc ctg ggacag ggt 2832 Gln Asp Arg Arg Leu Ala Ser Asp Asp Glu Arg Gly Leu Gly GlnGly 930 935 940 gtt ttg gac aac aag ccg gtg ctg cat att tat cgg ctg gtgctg gag 2880 Val Leu Asp Asn Lys Pro Val Leu His Ile Tyr Arg Leu Val LeuGlu 945 950 955 960 aag gtt aac aac tgt gtc cga ccg tca aag ctt cat cctgcc ggc tat 2928 Lys Val Asn Asn Cys Val Arg Pro Ser Lys Leu His Pro AlaGly Tyr 965 970 975 ttg aca agt gcc gca cac aaa gca tcg cag tca ctg ctggat cca ctg 2976 Leu Thr Ser Ala Ala His Lys Ala Ser Gln Ser Leu Leu AspPro Leu 980 985 990 gac aag ttt ata ttc gct gaa aat gag tgg atc ggg gcacag ggg caa 3024 Asp Lys Phe Ile Phe Ala Glu Asn Glu Trp Ile Gly Ala GlnGly Gln 995 1000 1005 ttt ggt ggc gat cat cct tcg gct cgt gag gat ctcgat gtg tcg gtg 3072 Phe Gly Gly Asp His Pro Ser Ala Arg Glu Asp Leu AspVal Ser Val 1010 1015 1020 atg aga cgc tta acc aag agc tcg gcc aaa acccag cga gta ggc tac 3120 Met Arg Arg Leu Thr Lys Ser Ser Ala Lys Thr GlnArg Val Gly Tyr 1025 1030 1035 1040 gtt ctg cac cgc acc aat ctg atg caatgc ggc act cca gag gag cat 3168 Val Leu His Arg Thr Asn Leu Met Gln CysGly Thr Pro Glu Glu His 1045 1050 1055 aca cag aag ctg gat gtg tgc caccta ctg ccg aat gtg gcg aga tgc 3216 Thr Gln Lys Leu Asp Val Cys His LeuLeu Pro Asn Val Ala Arg Cys 1060 1065 1070 gag cgc acg acg ctg act ttcctg cag aat ttg gag cac ttg gat ggc 3264 Glu Arg Thr Thr Leu Thr Phe LeuGln Asn Leu Glu His Leu Asp Gly 1075 1080 1085 atg gtg gcg ccg gaa gtgtgc ccc atg gaa acc gcc gct tat gtg agc 3312 Met Val Ala Pro Glu Val CysPro Met Glu Thr Ala Ala Tyr Val Ser 1090 1095 1100 agt cac tca agc tga3327 Ser His Ser Ser 1105 53 3435 DNA Homo sapiens CDS (1)..(3432) 53atg aag tta agc cgc cag ttc acc gtg ttc ggc agt gcg atc ttc tgt 48 MetLys Leu Ser Arg Gln Phe Thr Val Phe Gly Ser Ala Ile Phe Cys 1 5 10 15gtg gtg att ttc tcg ctc tac ctg atg ctg gac cgg ggt cac tta gac 96 ValVal Ile Phe Ser Leu Tyr Leu Met Leu Asp Arg Gly His Leu Asp 20 25 30 tacccc agg aac ccg cgc cgc gag ggc tcc ttc cct cag ggc cag ctc 144 Tyr ProArg Asn Pro Arg Arg Glu Gly Ser Phe Pro Gln Gly Gln Leu 35 40 45 tca atgttg caa gaa aaa ata gac cat ttg gag cgt ttg cta gct gag 192 Ser Met LeuGln Glu Lys Ile Asp His Leu Glu Arg Leu Leu Ala Glu 50 55 60 aat aat gagatc atc tca aat att aga gac tca gtc atc aat ttg agt 240 Asn Asn Glu IleIle Ser Asn Ile Arg Asp Ser Val Ile Asn Leu Ser 65 70 75 80 gag tct gtggag gat ggt ccg aaa agt tca caa agc aat ttc agc caa 288 Glu Ser Val GluAsp Gly Pro Lys Ser Ser Gln Ser Asn Phe Ser Gln 85 90 95 ggt gct ggc tcacat ctt ctg ccc tca caa tta tcc ctc tca gtt gac 336 Gly Ala Gly Ser HisLeu Leu Pro Ser Gln Leu Ser Leu Ser Val Asp 100 105 110 act gca gac tgtctg ttt gct tca caa agt gga agt cac aat tca gat 384 Thr Ala Asp Cys LeuPhe Ala Ser Gln Ser Gly Ser His Asn Ser Asp 115 120 125 gtg cag atg ttggat gtt tac agt cta att tct ttt gac aat cca gat 432 Val Gln Met Leu AspVal Tyr Ser Leu Ile Ser Phe Asp Asn Pro Asp 130 135 140 ggt gga gtt tggaag caa gga ttt gac att act tat gaa tct aat gaa 480 Gly Gly Val Trp LysGln Gly Phe Asp Ile Thr Tyr Glu Ser Asn Glu 145 150 155 160 tgg gac actgaa ccc ctt caa gtc ttt gtg gtg cct cat tcc cat aac 528 Trp Asp Thr GluPro Leu Gln Val Phe Val Val Pro His Ser His Asn 165 170 175 gac cca ggttgg ttg aag act ttc aat gac tac ttt aga gac aag act 576 Asp Pro Gly TrpLeu Lys Thr Phe Asn Asp Tyr Phe Arg Asp Lys Thr 180 185 190 cag tat attttt aat aac atg gtc cta aag ctg aaa gaa gac tca cgg 624 Gln Tyr Ile PheAsn Asn Met Val Leu Lys Leu Lys Glu Asp Ser Arg 195 200 205 agg aag tttatt tgg tct gag atc tct tac ctt tca aag tgg tgg gat 672 Arg Lys Phe IleTrp Ser Glu Ile Ser Tyr Leu Ser Lys Trp Trp Asp 210 215 220 att ata gatatt cag aag aag gat gct gtt aaa agt tta ata gaa aat 720 Ile Ile Asp IleGln Lys Lys Asp Ala Val Lys Ser Leu Ile Glu Asn 225 230 235 240 ggt cagctt gaa att gtg aca ggt ggc tgg gtt atg cct gat gaa gct 768 Gly Gln LeuGlu Ile Val Thr Gly Gly Trp Val Met Pro Asp Glu Ala 245 250 255 act ccacat tat ttt gcc tta att gat caa cta att gaa gga cat cag 816 Thr Pro HisTyr Phe Ala Leu Ile Asp Gln Leu Ile Glu Gly His Gln 260 265 270 tgg ctggaa aat aat ata gga gtg aaa cct cgg tcc ggc tgg gct att 864 Trp Leu GluAsn Asn Ile Gly Val Lys Pro Arg Ser Gly Trp Ala Ile 275 280 285 gat cccttt gga cac tca cca aca atg gct tat ctt cta aac cgt gct 912 Asp Pro PheGly His Ser Pro Thr Met Ala Tyr Leu Leu Asn Arg Ala 290 295 300 gga ctttct cac atg ctt atc cag aga gtt cat tat gca gtt aaa aaa 960 Gly Leu SerHis Met Leu Ile Gln Arg Val His Tyr Ala Val Lys Lys 305 310 315 320 cacttt gca ctg cat aaa aca ttg gag ttt ttt tgg aga cag aat tgg 1008 His PheAla Leu His Lys Thr Leu Glu Phe Phe Trp Arg Gln Asn Trp 325 330 335 gatctg gga tct gtc aca gat att tta tgc cac atg atg ccc ttc tac 1056 Asp LeuGly Ser Val Thr Asp Ile Leu Cys His Met Met Pro Phe Tyr 340 345 350 agctat gac atc cct cac act tgt gga cct gat cct aaa ata tgc tgc 1104 Ser TyrAsp Ile Pro His Thr Cys Gly Pro Asp Pro Lys Ile Cys Cys 355 360 365 cagttt gat ttt aaa cgt ctt cct gga ggc aga ttt ggt tgt ccc tgg 1152 Gln PheAsp Phe Lys Arg Leu Pro Gly Gly Arg Phe Gly Cys Pro Trp 370 375 380 ggagtc ccc cca gaa aca ata cat cct gga aat gtc caa agc agg gct 1200 Gly ValPro Pro Glu Thr Ile His Pro Gly Asn Val Gln Ser Arg Ala 385 390 395 400cgg atg cta cta gat cag tac cga aag aag tca aag ctt ttt cga acc 1248 ArgMet Leu Leu Asp Gln Tyr Arg Lys Lys Ser Lys Leu Phe Arg Thr 405 410 415aaa gtt ctc ctg gct cca cta gga gat gat ttc cgc tac tgt gaa tac 1296 LysVal Leu Leu Ala Pro Leu Gly Asp Asp Phe Arg Tyr Cys Glu Tyr 420 425 430acg gaa tgg gat tta cag ttt aag aat tat cag cag ctt ttt gat tat 1344 ThrGlu Trp Asp Leu Gln Phe Lys Asn Tyr Gln Gln Leu Phe Asp Tyr 435 440 445atg aat tct cag tcc aag ttt aaa gtt aag ata cag ttt gga act tta 1392 MetAsn Ser Gln Ser Lys Phe Lys Val Lys Ile Gln Phe Gly Thr Leu 450 455 460tca gat ttt ttt gat gcg ctg gat aaa gca gat gaa act cag aga gac 1440 SerAsp Phe Phe Asp Ala Leu Asp Lys Ala Asp Glu Thr Gln Arg Asp 465 470 475480 aag ggc caa tcg atg ttc cct gtt tta agt gga gat ttt ttc act tat 1488Lys Gly Gln Ser Met Phe Pro Val Leu Ser Gly Asp Phe Phe Thr Tyr 485 490495 gcc gat cga gat gat cat tac tgg agt ggc tat ttt aca tcc aga ccc 1536Ala Asp Arg Asp Asp His Tyr Trp Ser Gly Tyr Phe Thr Ser Arg Pro 500 505510 ttt tac aaa cga atg gac aga atc atg gaa tct cat tta agg gct gct 1584Phe Tyr Lys Arg Met Asp Arg Ile Met Glu Ser His Leu Arg Ala Ala 515 520525 gaa att ctt tac tat ttc gcc ctg aga caa gct cac aaa tac aag ata 1632Glu Ile Leu Tyr Tyr Phe Ala Leu Arg Gln Ala His Lys Tyr Lys Ile 530 535540 aat aaa ttt ctc tca tca tca ctt tac acg gca ctg aca gaa gcc aga 1680Asn Lys Phe Leu Ser Ser Ser Leu Tyr Thr Ala Leu Thr Glu Ala Arg 545 550555 560 agg aat ttg gga ctg ttt caa cat cat gat gct atc aca gga act gca1728 Arg Asn Leu Gly Leu Phe Gln His His Asp Ala Ile Thr Gly Thr Ala 565570 575 aaa gac tgg gtg gtt gtg gat tat ggt acc aga ctt ttt cat tcg tta1776 Lys Asp Trp Val Val Val Asp Tyr Gly Thr Arg Leu Phe His Ser Leu 580585 590 atg gtt ttg gag aag ata att gga aat tct gca ttt ctt ctt att ggg1824 Met Val Leu Glu Lys Ile Ile Gly Asn Ser Ala Phe Leu Leu Ile Gly 595600 605 aag gac aaa ctc aca tac gac tct tac tct cct gat acc ttc ctg gag1872 Lys Asp Lys Leu Thr Tyr Asp Ser Tyr Ser Pro Asp Thr Phe Leu Glu 610615 620 atg gat ttg aaa caa aaa tca caa gat tct ctg cca caa aaa aat ata1920 Met Asp Leu Lys Gln Lys Ser Gln Asp Ser Leu Pro Gln Lys Asn Ile 625630 635 640 ata agg ctg agt gcg gag cca agg tac ctt gtg gtc tat aat ccttta 1968 Ile Arg Leu Ser Ala Glu Pro Arg Tyr Leu Val Val Tyr Asn Pro Leu645 650 655 gaa caa gac cga atc tcg ttg gtc tca gtc tat gtg agt tcc ccgaca 2016 Glu Gln Asp Arg Ile Ser Leu Val Ser Val Tyr Val Ser Ser Pro Thr660 665 670 gtg caa gtg ttc tct gct tca gga aaa cct gtg gaa gtt caa gtcagc 2064 Val Gln Val Phe Ser Ala Ser Gly Lys Pro Val Glu Val Gln Val Ser675 680 685 gca gtt tgg gat aca gca aat act att tca gaa aca gcc tat gagatc 2112 Ala Val Trp Asp Thr Ala Asn Thr Ile Ser Glu Thr Ala Tyr Glu Ile690 695 700 tct ttt cga gca cat ata ccg cca ttg gga ctg aaa gtg tat aagatt 2160 Ser Phe Arg Ala His Ile Pro Pro Leu Gly Leu Lys Val Tyr Lys Ile705 710 715 720 ttg gaa tca gca agt tca aat tca cat tta gct gat tat gtcttg tat 2208 Leu Glu Ser Ala Ser Ser Asn Ser His Leu Ala Asp Tyr Val LeuTyr 725 730 735 aag aat aaa gta gaa gat agc gga att ttc acc ata aag aatatg ata 2256 Lys Asn Lys Val Glu Asp Ser Gly Ile Phe Thr Ile Lys Asn MetIle 740 745 750 aat act gaa gaa ggt ata aca cta gag aac tcc ttt gtt ttactt cgg 2304 Asn Thr Glu Glu Gly Ile Thr Leu Glu Asn Ser Phe Val Leu LeuArg 755 760 765 ttt gat caa act gga ctt atg aag caa atg atg act aaa gaagat ggt 2352 Phe Asp Gln Thr Gly Leu Met Lys Gln Met Met Thr Lys Glu AspGly 770 775 780 aaa cac cat gaa gta aat gtg caa ttt tca tgg tat gga accaca att 2400 Lys His His Glu Val Asn Val Gln Phe Ser Trp Tyr Gly Thr ThrIle 785 790 795 800 aaa aga gac aaa agt ggt gcc tac ctc ttc tta cct gatggt aat gcc 2448 Lys Arg Asp Lys Ser Gly Ala Tyr Leu Phe Leu Pro Asp GlyAsn Ala 805 810 815 aag cct tat gtt tac aca aca ccg ccc ttt gtc aga gtgaca cat gga 2496 Lys Pro Tyr Val Tyr Thr Thr Pro Pro Phe Val Arg Val ThrHis Gly 820 825 830 agg att tat tcg gaa gtg act tgc ttt ttt gac cat gttact cat aga 2544 Arg Ile Tyr Ser Glu Val Thr Cys Phe Phe Asp His Val ThrHis Arg 835 840 845 gtc cga cta tac cac ata cag gga ata gaa gga cag tctgtg gaa gtt 2592 Val Arg Leu Tyr His Ile Gln Gly Ile Glu Gly Gln Ser ValGlu Val 850 855 860 tcc aat att gtg gac atc cga aaa gta tat aac cgt gagatt gca atg 2640 Ser Asn Ile Val Asp Ile Arg Lys Val Tyr Asn Arg Glu IleAla Met 865 870 875 880 aaa att tct tct gat ata aaa agc caa aat aga ttttat act gac cta 2688 Lys Ile Ser Ser Asp Ile Lys Ser Gln Asn Arg Phe TyrThr Asp Leu 885 890 895 aat ggg tac cag att caa cct aga atg aca ctg agcaaa ttg cct ctt 2736 Asn Gly Tyr Gln Ile Gln Pro Arg Met Thr Leu Ser LysLeu Pro Leu 900 905 910 caa gca aat gtc tat ccc atg acc aca atg gcc tatatc cag gat gcc 2784 Gln Ala Asn Val Tyr Pro Met Thr Thr Met Ala Tyr IleGln Asp Ala 915 920 925 aaa cat cgt ttg aca ctg ctc tct gct cag tca ttaggg gtt tcg agt 2832 Lys His Arg Leu Thr Leu Leu Ser Ala Gln Ser Leu GlyVal Ser Ser 930 935 940 ttg aat agt ggt cag att gaa gtt atc atg gat cgaaga ctc atg caa 2880 Leu Asn Ser Gly Gln Ile Glu Val Ile Met Asp Arg ArgLeu Met Gln 945 950 955 960 gat gat aat cgt ggc ctt gag caa ggt atc caggat aac aag att aca 2928 Asp Asp Asn Arg Gly Leu Glu Gln Gly Ile Gln AspAsn Lys Ile Thr 965 970 975 gct aat cta ttt cga ata cta cta gaa aaa agaagt gct gtt aat acg 2976 Ala Asn Leu Phe Arg Ile Leu Leu Glu Lys Arg SerAla Val Asn Thr 980 985 990 gaa gaa gaa aag aag tcg gtc agt tat cct tctctc ctt agc cac ata 3024 Glu Glu Glu Lys Lys Ser Val Ser Tyr Pro Ser LeuLeu Ser His Ile 995 1000 1005 act tct tct ctc atg aat cat cca gtc attcca atg gca aat aag ttc 3072 Thr Ser Ser Leu Met Asn His Pro Val Ile ProMet Ala Asn Lys Phe 1010 1015 1020 tcc tca cct acc ctt gag ctg caa ggtgaa ttc tct cca tta cag tca 3120 Ser Ser Pro Thr Leu Glu Leu Gln Gly GluPhe Ser Pro Leu Gln Ser 1025 1030 1035 1040 tct ttg cct tgt gac att catctg gtt aat ttg aga aca ata cag tca 3168 Ser Leu Pro Cys Asp Ile His LeuVal Asn Leu Arg Thr Ile Gln Ser 1045 1050 1055 aag gtg ggc aat ggg cactcc aat gag gca gcc ttg atc ctc cac aga 3216 Lys Val Gly Asn Gly His SerAsn Glu Ala Ala Leu Ile Leu His Arg 1060 1065 1070 aaa ggg ttt gat tgtcgg ttc tct agc aaa ggc aca ggg ctg ttt tgt 3264 Lys Gly Phe Asp Cys ArgPhe Ser Ser Lys Gly Thr Gly Leu Phe Cys 1075 1080 1085 tct act act caggga aag ata ttg gta cag aaa ctt tta aac aag ttt 3312 Ser Thr Thr Gln GlyLys Ile Leu Val Gln Lys Leu Leu Asn Lys Phe 1090 1095 1100 att gtc gaaagt ctc aca cct tca tca cta tcc ttg atg cat tca cct 3360 Ile Val Glu SerLeu Thr Pro Ser Ser Leu Ser Leu Met His Ser Pro 1105 1110 1115 1120 cccggc act cag aat ata agt gag atc aac ttg agt cca atg gaa atc 3408 Pro GlyThr Gln Asn Ile Ser Glu Ile Asn Leu Ser Pro Met Glu Ile 1125 1130 1135agc aca ttc cga atc cag ttg agg tga 3435 Ser Thr Phe Arg Ile Gln Leu Arg1140 54 3453 DNA Mus musculus CDS (1)..(3450) 54 atg aag tta agt cgc cagttc acc gtg ttt ggc agc gcg atc ttc tgc 48 Met Lys Leu Ser Arg Gln PheThr Val Phe Gly Ser Ala Ile Phe Cys 1 5 10 15 gtc gta atc ttc tca ctctac ctg atg ctg gac agg ggt cac ttg gac 96 Val Val Ile Phe Ser Leu TyrLeu Met Leu Asp Arg Gly His Leu Asp 20 25 30 tac cct cgg ggc ccg cgc caggag ggc tcc ttt ccg cag ggc cag ctt 144 Tyr Pro Arg Gly Pro Arg Gln GluGly Ser Phe Pro Gln Gly Gln Leu 35 40 45 tca ata ttg caa gaa aag att gaccat ttg gag cgt ttg ctc gct gag 192 Ser Ile Leu Gln Glu Lys Ile Asp HisLeu Glu Arg Leu Leu Ala Glu 50 55 60 aac aac gag atc atc tca aat atc agagac tca gtc atc aac ctg agc 240 Asn Asn Glu Ile Ile Ser Asn Ile Arg AspSer Val Ile Asn Leu Ser 65 70 75 80 gag tct gtg gag gac ggc ccg cgg gggtca cca ggc aac gcc agc caa 288 Glu Ser Val Glu Asp Gly Pro Arg Gly SerPro Gly Asn Ala Ser Gln 85 90 95 ggc tcc atc cac ctc cac tcg cca cag ttggcc ctg cag gct gac ccc 336 Gly Ser Ile His Leu His Ser Pro Gln Leu AlaLeu Gln Ala Asp Pro 100 105 110 aga gac tgt ttg ttt gct tca cag agt gggagt cag ccc cgg gat gtg 384 Arg Asp Cys Leu Phe Ala Ser Gln Ser Gly SerGln Pro Arg Asp Val 115 120 125 cag atg ttg gat gtt tac gat ctg att cctttt gat aat cca gat ggt 432 Gln Met Leu Asp Val Tyr Asp Leu Ile Pro PheAsp Asn Pro Asp Gly 130 135 140 gga gtt tgg aag caa gga ttt gac att aagtat gaa gcg gat gag tgg 480 Gly Val Trp Lys Gln Gly Phe Asp Ile Lys TyrGlu Ala Asp Glu Trp 145 150 155 160 gac cat gag ccc ctg caa gtg ttt gtggtg cct cac tcc cat aat gac 528 Asp His Glu Pro Leu Gln Val Phe Val ValPro His Ser His Asn Asp 165 170 175 cca ggt tgg ttg aag act ttc aat gactac ttt aga gac aag act cag 576 Pro Gly Trp Leu Lys Thr Phe Asn Asp TyrPhe Arg Asp Lys Thr Gln 180 185 190 tat att ttt aat aac atg gtc cta aagctg aaa gaa gac tca agc agg 624 Tyr Ile Phe Asn Asn Met Val Leu Lys LeuLys Glu Asp Ser Ser Arg 195 200 205 aag ttt atg tgg tct gag atc tct tacctt gca aaa tgg tgg gat att 672 Lys Phe Met Trp Ser Glu Ile Ser Tyr LeuAla Lys Trp Trp Asp Ile 210 215 220 ata gat att ccg aag aag gaa gct gttaaa agt tta cta cag aat ggt 720 Ile Asp Ile Pro Lys Lys Glu Ala Val LysSer Leu Leu Gln Asn Gly 225 230 235 240 cag ctg gaa att gtg acc ggt ggctgg gtt atg cct gat gaa gcc act 768 Gln Leu Glu Ile Val Thr Gly Gly TrpVal Met Pro Asp Glu Ala Thr 245 250 255 cca cat tat ttt gcc tta att gaccaa cta att gaa ggg cac caa tgg 816 Pro His Tyr Phe Ala Leu Ile Asp GlnLeu Ile Glu Gly His Gln Trp 260 265 270 ctg gaa aaa aat cta gga gtg aaacct cga tcg ggc tgg gcc ata gat 864 Leu Glu Lys Asn Leu Gly Val Lys ProArg Ser Gly Trp Ala Ile Asp 275 280 285 ccc ttt ggt cat tca ccc aca atggct tat ctt cta aag cgt gct gga 912 Pro Phe Gly His Ser Pro Thr Met AlaTyr Leu Leu Lys Arg Ala Gly 290 295 300 ttt tca cac atg ctc atc cag agagtc cat tat gca atc aaa aaa cac 960 Phe Ser His Met Leu Ile Gln Arg ValHis Tyr Ala Ile Lys Lys His 305 310 315 320 ttc tct ttg cat aaa acg ctggag ttt ttc tgg aga cag aat tgg gat 1008 Phe Ser Leu His Lys Thr Leu GluPhe Phe Trp Arg Gln Asn Trp Asp 325 330 335 ctt gga tct gct aca gac attttg tgc cat atg atg ccc ttc tac agc 1056 Leu Gly Ser Ala Thr Asp Ile LeuCys His Met Met Pro Phe Tyr Ser 340 345 350 tac gac atc cct cac acc tgtggg cct gat cct aaa ata tgc tgc cag 1104 Tyr Asp Ile Pro His Thr Cys GlyPro Asp Pro Lys Ile Cys Cys Gln 355 360 365 ttt gat ttt aaa cgg ctt cctgga ggc aga tat ggt tgt ccc tgg gga 1152 Phe Asp Phe Lys Arg Leu Pro GlyGly Arg Tyr Gly Cys Pro Trp Gly 370 375 380 gtt ccc cca gaa gca ata tctcct gga aat gtc caa agc agg gct cag 1200 Val Pro Pro Glu Ala Ile Ser ProGly Asn Val Gln Ser Arg Ala Gln 385 390 395 400 atg cta ttg gat cag taccgg aaa aag tca aaa ctt ttc cgc act aaa 1248 Met Leu Leu Asp Gln Tyr ArgLys Lys Ser Lys Leu Phe Arg Thr Lys 405 410 415 gtt ctg ctg gct cca ctggga gac gac ttt cgg ttc agt gaa tac aca 1296 Val Leu Leu Ala Pro Leu GlyAsp Asp Phe Arg Phe Ser Glu Tyr Thr 420 425 430 gag tgg gat ctg cag tgcagg aac tac gag caa ctg ttc agt tac atg 1344 Glu Trp Asp Leu Gln Cys ArgAsn Tyr Glu Gln Leu Phe Ser Tyr Met 435 440 445 aac tcg cag cct cat ctgaaa gtg aag atc cag ttt gga acc ttg tca 1392 Asn Ser Gln Pro His Leu LysVal Lys Ile Gln Phe Gly Thr Leu Ser 450 455 460 gat tat ttc gac gca ttggag aaa gcg gtg gca gcc gag aag aag agt 1440 Asp Tyr Phe Asp Ala Leu GluLys Ala Val Ala Ala Glu Lys Lys Ser 465 470 475 480 agc cag tct gtg ttccct gcc ctg agt gga gac ttc ttc acg tac gct 1488 Ser Gln Ser Val Phe ProAla Leu Ser Gly Asp Phe Phe Thr Tyr Ala 485 490 495 gac aga gac gac cattac tgg agt ggc tac ttc acg tcc aga cct ttc 1536 Asp Arg Asp Asp His TyrTrp Ser Gly Tyr Phe Thr Ser Arg Pro Phe 500 505 510 tac aaa cga atg gacaga ata atg gaa tct cgt ata agg gct gct gaa 1584 Tyr Lys Arg Met Asp ArgIle Met Glu Ser Arg Ile Arg Ala Ala Glu 515 520 525 att ctt tac cag ttggcc ttg aaa caa gct cag aaa tac aag ata aat 1632 Ile Leu Tyr Gln Leu AlaLeu Lys Gln Ala Gln Lys Tyr Lys Ile Asn 530 535 540 aaa ttt ctt tca tcacct cat tac aca aca ctg aca gaa gcc aga agg 1680 Lys Phe Leu Ser Ser ProHis Tyr Thr Thr Leu Thr Glu Ala Arg Arg 545 550 555 560 aac tta gga ctattt cag cat cat gat gcc atc aca gga acc gcg aaa 1728 Asn Leu Gly Leu PheGln His His Asp Ala Ile Thr Gly Thr Ala Lys 565 570 575 gac tgg gtg gttgtg gac tat ggt acc aga ctc ttt cag tca tta aat 1776 Asp Trp Val Val ValAsp Tyr Gly Thr Arg Leu Phe Gln Ser Leu Asn 580 585 590 tct ttg gag aagata att gga gat tct gca ttt ctt ctc att tta aag 1824 Ser Leu Glu Lys IleIle Gly Asp Ser Ala Phe Leu Leu Ile Leu Lys 595 600 605 gac aaa aag ctgtac cag tca gat cct tcc aaa gcc ttc tta gag atg 1872 Asp Lys Lys Leu TyrGln Ser Asp Pro Ser Lys Ala Phe Leu Glu Met 610 615 620 gat acg aag caaagt tca caa gat tct ctg ccc caa aaa att ata ata 1920 Asp Thr Lys Gln SerSer Gln Asp Ser Leu Pro Gln Lys Ile Ile Ile 625 630 635 640 caa ctg agcgca cag gag cca agg tac ctt gtg gtc tac aat ccc ttt 1968 Gln Leu Ser AlaGln Glu Pro Arg Tyr Leu Val Val Tyr Asn Pro Phe 645 650 655 gaa caa gaacgg cat tca gtg gtg tcc atc cgg gta aac tcc gcc aca 2016 Glu Gln Glu ArgHis Ser Val Val Ser Ile Arg Val Asn Ser Ala Thr 660 665 670 ggg aaa gtgctg tct gat tcg gga aaa ccg gtg gag gtt caa gtc agt 2064 Gly Lys Val LeuSer Asp Ser Gly Lys Pro Val Glu Val Gln Val Ser 675 680 685 gca gtt tggaac gac atg agg aca att tca caa gca gcc tat gag gtt 2112 Ala Val Trp AsnAsp Met Arg Thr Ile Ser Gln Ala Ala Tyr Glu Val 690 695 700 tct ttt ctagct cat ata cca cca ctg gga ctg aaa gtg ttt aag atc 2160 Ser Phe Leu AlaHis Ile Pro Pro Leu Gly Leu Lys Val Phe Lys Ile 705 710 715 720 tta gagtca caa agt tca agc tca cac ttg gct gat tat gtc cta tat 2208 Leu Glu SerGln Ser Ser Ser Ser His Leu Ala Asp Tyr Val Leu Tyr 725 730 735 aat aatgat gga cta gca gaa aat gga ata ttc cac gtg aag aac atg 2256 Asn Asn AspGly Leu Ala Glu Asn Gly Ile Phe His Val Lys Asn Met 740 745 750 gtg gatgct gga gat gcc ata aca ata gag aat ccc ttc ctg gcg att 2304 Val Asp AlaGly Asp Ala Ile Thr Ile Glu Asn Pro Phe Leu Ala Ile 755 760 765 tgg tttgac cga tct ggg ctg atg gag aaa gtg aga agg aaa gaa gac 2352 Trp Phe AspArg Ser Gly Leu Met Glu Lys Val Arg Arg Lys Glu Asp 770 775 780 agt agacag cat gaa ctg aag gtc cag ttc ctg tgg tac gga acc acc 2400 Ser Arg GlnHis Glu Leu Lys Val Gln Phe Leu Trp Tyr Gly Thr Thr 785 790 795 800 aacaaa agg gac aag agc ggt gcc tac ctc ttc ctg cct gac ggg cag 2448 Asn LysArg Asp Lys Ser Gly Ala Tyr Leu Phe Leu Pro Asp Gly Gln 805 810 815 ggccag cca tat gtt tcc cta aga ccg ccc ttt gtc aga gtg aca cgt 2496 Gly GlnPro Tyr Val Ser Leu Arg Pro Pro Phe Val Arg Val Thr Arg 820 825 830 ggaagg atc tac tca gat gtg acc tgt ttc ctc gaa cac gtt act cac 2544 Gly ArgIle Tyr Ser Asp Val Thr Cys Phe Leu Glu His Val Thr His 835 840 845 aaagtc cgc ctg tac aac att cag gga ata gaa ggt cag tcc atg gaa 2592 Lys ValArg Leu Tyr Asn Ile Gln Gly Ile Glu Gly Gln Ser Met Glu 850 855 860 gtttct aat att gta aac atc agg aat gtg cat aac cgt gag att gta 2640 Val SerAsn Ile Val Asn Ile Arg Asn Val His Asn Arg Glu Ile Val 865 870 875 880atg aga att tca tct aaa ata aac aac caa aat aga tat tat act gac 2688 MetArg Ile Ser Ser Lys Ile Asn Asn Gln Asn Arg Tyr Tyr Thr Asp 885 890 895cta aat gga tat cag att cag cct aga agg acc atg agc aaa ttg cct 2736 LeuAsn Gly Tyr Gln Ile Gln Pro Arg Arg Thr Met Ser Lys Leu Pro 900 905 910ctt caa gcc aac gtt tac ccg atg tgc aca atg gcg tat atc cag gat 2784 LeuGln Ala Asn Val Tyr Pro Met Cys Thr Met Ala Tyr Ile Gln Asp 915 920 925gct gag cac cgg ctc acg ctg ctc tct gct cag tct cta ggt gct tcc 2832 AlaGlu His Arg Leu Thr Leu Leu Ser Ala Gln Ser Leu Gly Ala Ser 930 935 940agc atg gct tct ggt cag att gaa gtc ttc atg gat cga agg ctc atg 2880 SerMet Ala Ser Gly Gln Ile Glu Val Phe Met Asp Arg Arg Leu Met 945 950 955960 cag gat gat aac cgt ggc ctt ggg caa ggc gtc cat gac aat aag att 2928Gln Asp Asp Asn Arg Gly Leu Gly Gln Gly Val His Asp Asn Lys Ile 965 970975 aca gct aat ttg ttt cga atc ctc ctc gag aag aga agc gct gtg aac 2976Thr Ala Asn Leu Phe Arg Ile Leu Leu Glu Lys Arg Ser Ala Val Asn 980 985990 atg gaa gaa gaa aag aag agc cct gtc agc tac cct tcc ctc ctc agc 3024Met Glu Glu Glu Lys Lys Ser Pro Val Ser Tyr Pro Ser Leu Leu Ser 995 10001005 cac atg act tcg tcc ttc ctc aac cat ccc ttt ctc ccc atg gta cta3072 His Met Thr Ser Ser Phe Leu Asn His Pro Phe Leu Pro Met Val Leu1010 1015 1020 agt ggc cag ctc ccc tcc cct gcc ttt gag ctg ctg agt gaattt cct 3120 Ser Gly Gln Leu Pro Ser Pro Ala Phe Glu Leu Leu Ser Glu PhePro 1025 1030 1035 1040 ctg ctg cag tcc tct cta cct tgt gat atc cat ctggtc aac ctg cgg 3168 Leu Leu Gln Ser Ser Leu Pro Cys Asp Ile His Leu ValAsn Leu Arg 1045 1050 1055 aca ata caa tca aag atg ggc aaa ggc tat tcggat gag gca gcc ttg 3216 Thr Ile Gln Ser Lys Met Gly Lys Gly Tyr Ser AspGlu Ala Ala Leu 1060 1065 1070 atc ctc cac agg aaa ggg ttt gat tgc cagttc tcc agc aga ggc atc 3264 Ile Leu His Arg Lys Gly Phe Asp Cys Gln PheSer Ser Arg Gly Ile 1075 1080 1085 ggg cta ccc tgt tcc act act cag ggaaag atg tca gtt ctg aaa ctt 3312 Gly Leu Pro Cys Ser Thr Thr Gln Gly LysMet Ser Val Leu Lys Leu 1090 1095 1100 ttc aac aag ttt gct gtg gag agtctc gtc cct tcc tct ctg tcc ttg 3360 Phe Asn Lys Phe Ala Val Glu Ser LeuVal Pro Ser Ser Leu Ser Leu 1105 1110 1115 1120 atg cac tcc cct cca gatgcc cag aac atg agt gaa gtc agc ctg agc 3408 Met His Ser Pro Pro Asp AlaGln Asn Met Ser Glu Val Ser Leu Ser 1125 1130 1135 ccc atg gag atc agcacg ttc cgt atc cgc ttg cgt tgg acc tga 3453 Pro Met Glu Ile Ser Thr PheArg Ile Arg Leu Arg Trp Thr 1140 1145 1150 55 3840 DNA Rattus norvegicusCDS (1)..(3837) 55 atg gcc tgt ata ggt gga gcc cag ggg caa cgg cag gccgtg gaa aag 48 Met Ala Cys Ile Gly Gly Ala Gln Gly Gln Arg Gln Ala ValGlu Lys 1 5 10 15 gaa cct tcc cac caa ggg tat ccg tgg aag cca atg accaat ggc agc 96 Glu Pro Ser His Gln Gly Tyr Pro Trp Lys Pro Met Thr AsnGly Ser 20 25 30 tgc tca gaa ctg gca ttg ctc agc aaa acc cga atg tac tgtcac cag 144 Cys Ser Glu Leu Ala Leu Leu Ser Lys Thr Arg Met Tyr Cys HisGln 35 40 45 gga tgt gtc agg cca ccc agg act gac gtg aaa aac ttc aag accaca 192 Gly Cys Val Arg Pro Pro Arg Thr Asp Val Lys Asn Phe Lys Thr Thr50 55 60 act gat act cag agt gtg cct ggt gtc agt atg aag ctg aaa aag cag240 Thr Asp Thr Gln Ser Val Pro Gly Val Ser Met Lys Leu Lys Lys Gln 6570 75 80 gtg aca gtg tgc ggg gct gct atc ttc tgt gtg gcc gtc ttt tcc ctg288 Val Thr Val Cys Gly Ala Ala Ile Phe Cys Val Ala Val Phe Ser Leu 8590 95 tac cta atg ctg gac cga gtg cag cat gat cct gcc aga cac cag aat336 Tyr Leu Met Leu Asp Arg Val Gln His Asp Pro Ala Arg His Gln Asn 100105 110 ggt ggg aac ttc ccc agg agc caa att tct gtg cta cag aac cgg atc384 Gly Gly Asn Phe Pro Arg Ser Gln Ile Ser Val Leu Gln Asn Arg Ile 115120 125 gaa cag ctg gaa cag ctg ctg gaa gaa aac cat gag atc ata agc cat432 Glu Gln Leu Glu Gln Leu Leu Glu Glu Asn His Glu Ile Ile Ser His 130135 140 atc aag gac tct gtg ctg gaa ctg aca gcc aat gcg gag ggc cca cca480 Ile Lys Asp Ser Val Leu Glu Leu Thr Ala Asn Ala Glu Gly Pro Pro 145150 155 160 gcc ctg ctg ccc tac cac aca gcc aac ggc tcc tgg gct gtg ctcccc 528 Ala Leu Leu Pro Tyr His Thr Ala Asn Gly Ser Trp Ala Val Leu Pro165 170 175 gag ccc cgg ccc agc ttc ttc tct gta tcc cct gag gac tgc cagttt 576 Glu Pro Arg Pro Ser Phe Phe Ser Val Ser Pro Glu Asp Cys Gln Phe180 185 190 gct ttg ggg ggc cgg ggt cag aag cca gag cta cag atg tta actgtg 624 Ala Leu Gly Gly Arg Gly Gln Lys Pro Glu Leu Gln Met Leu Thr Val195 200 205 tct gag gat ttg ccg ttt gac aat gtg gag ggc ggc gtg tgg aggcaa 672 Ser Glu Asp Leu Pro Phe Asp Asn Val Glu Gly Gly Val Trp Arg Gln210 215 220 ggc ttc gac atc tcc tac agc cca aat gac tgg gat gct gaa gacctg 720 Gly Phe Asp Ile Ser Tyr Ser Pro Asn Asp Trp Asp Ala Glu Asp Leu225 230 235 240 cag gtg ttt gtg gtg cct cac tcc cac aat gat cca ggt gaagag cca 768 Gln Val Phe Val Val Pro His Ser His Asn Asp Pro Gly Glu GluPro 245 250 255 gca ggc ccc agc cgc agc gtg cag ggt ggg ctt tct ggt gacagg cgc 816 Ala Gly Pro Ser Arg Ser Val Gln Gly Gly Leu Ser Gly Asp ArgArg 260 265 270 tgg atc aag act ttt gac aag tac tac acg gaa caa acc caacac atc 864 Trp Ile Lys Thr Phe Asp Lys Tyr Tyr Thr Glu Gln Thr Gln HisIle 275 280 285 ctc aac agc atg gtg tcc aag ctg cag gaa gat ccc cga cggcgc ttt 912 Leu Asn Ser Met Val Ser Lys Leu Gln Glu Asp Pro Arg Arg ArgPhe 290 295 300 ctc tgg gca gaa gtc tcc ttc ttc gcc aag tgg tgg gac aacatc agt 960 Leu Trp Ala Glu Val Ser Phe Phe Ala Lys Trp Trp Asp Asn IleSer 305 310 315 320 gcc cag aaa agg gca gca gtt cga agg ctg gtg gga aatggg cag ctg 1008 Ala Gln Lys Arg Ala Ala Val Arg Arg Leu Val Gly Asn GlyGln Leu 325 330 335 gaa att gca acg ggt gga tgg gtg atg cca gat gag gccaac tcc cat 1056 Glu Ile Ala Thr Gly Gly Trp Val Met Pro Asp Glu Ala AsnSer His 340 345 350 tac ttt gcc ctg gtg ggg cag ctc atc gag ggg ccc cccccg gta cgc 1104 Tyr Phe Ala Leu Val Gly Gln Leu Ile Glu Gly Pro Pro ProVal Arg 355 360 365 agg gca gtg gac ccc ttt gga cac agc tcc acc atg ccttac ctg ctg 1152 Arg Ala Val Asp Pro Phe Gly His Ser Ser Thr Met Pro TyrLeu Leu 370 375 380 cgc cgt gcc aac ctg acc agc atg cta att cag agg gtgcat tac gcc 1200 Arg Arg Ala Asn Leu Thr Ser Met Leu Ile Gln Arg Val HisTyr Ala 385 390 395 400 atc aag aag cac ttt gcg gcc act cac agc ctg gagttc atg tgg agg 1248 Ile Lys Lys His Phe Ala Ala Thr His Ser Leu Glu PheMet Trp Arg 405 410 415 cag aca tgg gat tca gac tcc agc aca gac atc ttctgc cac atg atg 1296 Gln Thr Trp Asp Ser Asp Ser Ser Thr Asp Ile Phe CysHis Met Met 420 425 430 ccc ttc tac agc tac gac gtc cca cac acc tgt ggccct gat ccc aag 1344 Pro Phe Tyr Ser Tyr Asp Val Pro His Thr Cys Gly ProAsp Pro Lys 435 440 445 atc tgc tgc cag ttt gat ttc aaa cgt ctg ccg ggtggg aga atc aat 1392 Ile Cys Cys Gln Phe Asp Phe Lys Arg Leu Pro Gly GlyArg Ile Asn 450 455 460 tgt cct tgg aag gtg ccg ccg cgg gct atc aca gaggcc aac gtg gca 1440 Cys Pro Trp Lys Val Pro Pro Arg Ala Ile Thr Glu AlaAsn Val Ala 465 470 475 480 gac agg gca gcc ctg ctc ctg gac cag tac cggaag aag tcc cgg ctg 1488 Asp Arg Ala Ala Leu Leu Leu Asp Gln Tyr Arg LysLys Ser Arg Leu 485 490 495 ttt cga agc agt gtc ctc ctt gtg ccg ctg ggtgat gac ttc cga tat 1536 Phe Arg Ser Ser Val Leu Leu Val Pro Leu Gly AspAsp Phe Arg Tyr 500 505 510 gac aag ccc cag gaa tgg gat gcc cag ttc ttcaac tat caa cgg ctc 1584 Asp Lys Pro Gln Glu Trp Asp Ala Gln Phe Phe AsnTyr Gln Arg Leu 515 520 525 ttt gac ttc ctc aac agc aag ccg gag ttc cacgta cag gca cag ttt 1632 Phe Asp Phe Leu Asn Ser Lys Pro Glu Phe His ValGln Ala Gln Phe 530 535 540 ggg acc ctc tct gag tat ttt gat gcc ctg tataag agg aca gga gtg 1680 Gly Thr Leu Ser Glu Tyr Phe Asp Ala Leu Tyr LysArg Thr Gly Val 545 550 555 560 gag cct ggt gcc cgg cct cca ggg ttt cctgtg ctg agt ggg gac ttc 1728 Glu Pro Gly Ala Arg Pro Pro Gly Phe Pro ValLeu Ser Gly Asp Phe 565 570 575 ttc tcc tat gct gac cgg gag gac cac tactgg aca ggc tat tac act 1776 Phe Ser Tyr Ala Asp Arg Glu Asp His Tyr TrpThr Gly Tyr Tyr Thr 580 585 590 tcc cgg cct ttc tat aag agc ttg gac cgcgtg cta gaa act cac ctt 1824 Ser Arg Pro Phe Tyr Lys Ser Leu Asp Arg ValLeu Glu Thr His Leu 595 600 605 cgt ggg gca gag gtt cta tac agc ctg gctttg gcg cat gcc cgc cgt 1872 Arg Gly Ala Glu Val Leu Tyr Ser Leu Ala LeuAla His Ala Arg Arg 610 615 620 tct gga ctg act ggc cag tat ccg ctg tctgat tac gct gtc ctg acg 1920 Ser Gly Leu Thr Gly Gln Tyr Pro Leu Ser AspTyr Ala Val Leu Thr 625 630 635 640 gaa gct cgt cgt aca ctg ggc ctc ttccag cac cac gat gcc atc acc 1968 Glu Ala Arg Arg Thr Leu Gly Leu Phe GlnHis His Asp Ala Ile Thr 645 650 655 gga act gcc aag gag gca gtt gta gtagac tat ggg gtc agg ttg ctg 2016 Gly Thr Ala Lys Glu Ala Val Val Val AspTyr Gly Val Arg Leu Leu 660 665 670 cgt tcc ctg gtc agc cta aag cag gtcatc atc aat gct gcc cac tac 2064 Arg Ser Leu Val Ser Leu Lys Gln Val IleIle Asn Ala Ala His Tyr 675 680 685 ctg gtg ctg ggg gac aag gag acc tacagc ttt gac cct agg gca ccc 2112 Leu Val Leu Gly Asp Lys Glu Thr Tyr SerPhe Asp Pro Arg Ala Pro 690 695 700 ttc ctc caa atg gtg agc cag gcc tggcga ggc tct cag agc acc ctc 2160 Phe Leu Gln Met Val Ser Gln Ala Trp ArgGly Ser Gln Ser Thr Leu 705 710 715 720 cac ccc agc gcg gcc ctt gtt cctgct gct gct gct tct gcc ctg ctg 2208 His Pro Ser Ala Ala Leu Val Pro AlaAla Ala Ala Ser Ala Leu Leu 725 730 735 ccg cag cga gct cct agg ttt gtggtg gtc ttt aac cca ctg gaa cag 2256 Pro Gln Arg Ala Pro Arg Phe Val ValVal Phe Asn Pro Leu Glu Gln 740 745 750 gag cgg ctc agt gtg gtg tcc ctgctg gtc aac tca ccc cga gtg cga 2304 Glu Arg Leu Ser Val Val Ser Leu LeuVal Asn Ser Pro Arg Val Arg 755 760 765 gtg ctt tca gag gag ggt cag cccttg tct gtg cag atc agt gtg cag 2352 Val Leu Ser Glu Glu Gly Gln Pro LeuSer Val Gln Ile Ser Val Gln 770 775 780 tgg agc tcc gcc acc aac atg gtcccc gat gtc tac cag gtg tca gtg 2400 Trp Ser Ser Ala Thr Asn Met Val ProAsp Val Tyr Gln Val Ser Val 785 790 795 800 cct gtc cgc ctg cca gcc ctgggc ctg ggt gtg ctg cag ctg cag cca 2448 Pro Val Arg Leu Pro Ala Leu GlyLeu Gly Val Leu Gln Leu Gln Pro 805 810 815 gat ctc gat gga ccc tac acactg cag tct tcg gtg cat gtc tac ctg 2496 Asp Leu Asp Gly Pro Tyr Thr LeuGln Ser Ser Val His Val Tyr Leu 820 825 830 aac ggc gtg aaa ctg tct gtcagc agg caa aca aca ttc cct ctc cgt 2544 Asn Gly Val Lys Leu Ser Val SerArg Gln Thr Thr Phe Pro Leu Arg 835 840 845 gtt gtg gac tcg ggc acc agtgac ttc gcc atc agc aat cga tac atg 2592 Val Val Asp Ser Gly Thr Ser AspPhe Ala Ile Ser Asn Arg Tyr Met 850 855 860 cag gtc tgg ttc tcc ggc cttact ggg ctt ctc aag agc gtc cga cgt 2640 Gln Val Trp Phe Ser Gly Leu ThrGly Leu Leu Lys Ser Val Arg Arg 865 870 875 880 gtg gac gaa gag cag gaacag cag gtg gac atg aag ctc ttc gtc tat 2688 Val Asp Glu Glu Gln Glu GlnGln Val Asp Met Lys Leu Phe Val Tyr 885 890 895 gga acc cgc aca tcc aaggat aag agt ggt gcc tac ctc ttc ctg cct 2736 Gly Thr Arg Thr Ser Lys AspLys Ser Gly Ala Tyr Leu Phe Leu Pro 900 905 910 gat aac gag gct aag ccctat gtc cct aag aaa cct cct gtg ctg cgc 2784 Asp Asn Glu Ala Lys Pro TyrVal Pro Lys Lys Pro Pro Val Leu Arg 915 920 925 gtc acc gaa ggc cct ttcttc tca gag gtg gct gcg tat tat gag cac 2832 Val Thr Glu Gly Pro Phe PheSer Glu Val Ala Ala Tyr Tyr Glu His 930 935 940 ttt cac caa gtg att cgactt tac aac ctg cca ggg gta gag ggg ctg 2880 Phe His Gln Val Ile Arg LeuTyr Asn Leu Pro Gly Val Glu Gly Leu 945 950 955 960 tct ctg gac gtg tcgttc cag gtg gac atc agg gac tac gtg aac aag 2928 Ser Leu Asp Val Ser PheGln Val Asp Ile Arg Asp Tyr Val Asn Lys 965 970 975 gag cta gcc ctg cgcatc cac aca gac atc gac agc cag ggc act ttc 2976 Glu Leu Ala Leu Arg IleHis Thr Asp Ile Asp Ser Gln Gly Thr Phe 980 985 990 ttc aca gac ctc aatggc ttt cag gta cag ccc cgg aag tat ctg aag 3024 Phe Thr Asp Leu Asn GlyPhe Gln Val Gln Pro Arg Lys Tyr Leu Lys 995 1000 1005 aag ttg ccc ctgcag gct aat ttc tac cct atg cca gtc atg gcc tac 3072 Lys Leu Pro Leu GlnAla Asn Phe Tyr Pro Met Pro Val Met Ala Tyr 1010 1015 1020 atc cag gattcc cag agg cgc ctc acg ctg cac act gct cag gct ctg 3120 Ile Gln Asp SerGln Arg Arg Leu Thr Leu His Thr Ala Gln Ala Leu 1025 1030 1035 1040 ggtgtc tcc agc ctc ggc aat ggc cag ctg gag gtg atc ttg gac cga 3168 Gly ValSer Ser Leu Gly Asn Gly Gln Leu Glu Val Ile Leu Asp Arg 1045 1050 1055agg cta atg cag gat gac aac cgg gga cta ggc caa ggg ctc aaa gac 3216 ArgLeu Met Gln Asp Asp Asn Arg Gly Leu Gly Gln Gly Leu Lys Asp 1060 10651070 aac aag atc acc tgc aac cat ttc cgc ctc ctg tta gaa cgt cga acc3264 Asn Lys Ile Thr Cys Asn His Phe Arg Leu Leu Leu Glu Arg Arg Thr1075 1080 1085 ctg atg agc cct gag gtc caa cag gag cgc tct aca agc tacccg tcc 3312 Leu Met Ser Pro Glu Val Gln Gln Glu Arg Ser Thr Ser Tyr ProSer 1090 1095 1100 ctc ctc agc cac atg act tcc atg tac ctc aac aca cctcct ctg gtc 3360 Leu Leu Ser His Met Thr Ser Met Tyr Leu Asn Thr Pro ProLeu Val 1105 1110 1115 1120 tta ccg gtg gcc aag agg gag agc acc agc cccact ctg cac tct ttc 3408 Leu Pro Val Ala Lys Arg Glu Ser Thr Ser Pro ThrLeu His Ser Phe 1125 1130 1135 cac cct ctg gct tct ccg ttg ccc tgc gatttc cat ctg ctc aat ctg 3456 His Pro Leu Ala Ser Pro Leu Pro Cys Asp PheHis Leu Leu Asn Leu 1140 1145 1150 cgc atg ctc ccc gcc gag gtg agt gtcccg gtc cgt gcc aat cct cac 3504 Arg Met Leu Pro Ala Glu Val Ser Val ProVal Arg Ala Asn Pro His 1155 1160 1165 cat cag gct gag cct tgc ctt cttggc aga cat gct gct gac cct cca 3552 His Gln Ala Glu Pro Cys Leu Leu GlyArg His Ala Ala Asp Pro Pro 1170 1175 1180 ccg ctc ttg tcc ctg act gtcttc cag gac acc ttg ccc gcg gct gat 3600 Pro Leu Leu Ser Leu Thr Val PheGln Asp Thr Leu Pro Ala Ala Asp 1185 1190 1195 1200 gct gct ctc atc ctacac cgc aag ggt ttt gac tgt ggc ctt gaa gcc 3648 Ala Ala Leu Ile Leu HisArg Lys Gly Phe Asp Cys Gly Leu Glu Ala 1205 1210 1215 aag aac ctg ggcttc aac tgt acc aca agc caa ggc aag ctg gcc ctg 3696 Lys Asn Leu Gly PheAsn Cys Thr Thr Ser Gln Gly Lys Leu Ala Leu 1220 1225 1230 ggg agc ctcttc cat ggc ctg gat gtg cta ttc ctg cag ccc acc tct 3744 Gly Ser Leu PheHis Gly Leu Asp Val Leu Phe Leu Gln Pro Thr Ser 1235 1240 1245 ttg actttg cta tac cct ctg gcc tcg ccc tcc aac agc act gac atc 3792 Leu Thr LeuLeu Tyr Pro Leu Ala Ser Pro Ser Asn Ser Thr Asp Ile 1250 1255 1260 tctctg gag ccc atg gag atc agc acc ttc cgc ctg cgc ttg ggt tag 3840 Ser LeuGlu Pro Met Glu Ile Ser Thr Phe Arg Leu Arg Leu Gly 1265 1270 1275 563420 DNA Homo sapiens CDS (1)..(3417) 56 atg aag ctg aaa aag cag gtg acagtg tgt ggg gct gcc atc ttc tgt 48 Met Lys Leu Lys Lys Gln Val Thr ValCys Gly Ala Ala Ile Phe Cys 1 5 10 15 gtg gca gtc ttc tcg ctc tac ctcatg ctg gac cga gtg caa cac gat 96 Val Ala Val Phe Ser Leu Tyr Leu MetLeu Asp Arg Val Gln His Asp 20 25 30 ccc acc cga cac cag aat ggt ggg aacttc ccc cgg agc caa att tct 144 Pro Thr Arg His Gln Asn Gly Gly Asn PhePro Arg Ser Gln Ile Ser 35 40 45 gtg ctg cag aac cgc att gag cag ctg gagcag ctt ttg gag gag aac 192 Val Leu Gln Asn Arg Ile Glu Gln Leu Glu GlnLeu Leu Glu Glu Asn 50 55 60 cat gag att atc agc cat atc aag gac tcc gtgctg gag ctg aca gcc 240 His Glu Ile Ile Ser His Ile Lys Asp Ser Val LeuGlu Leu Thr Ala 65 70 75 80 aac gca gag ggc ccg ccc gcc atg ctg ccc tactac acg gtc aat ggc 288 Asn Ala Glu Gly Pro Pro Ala Met Leu Pro Tyr TyrThr Val Asn Gly 85 90 95 tcc tgg gtg gtg cca ccg gag ccc cgg ccc agc ttcttc tcc atc tcc 336 Ser Trp Val Val Pro Pro Glu Pro Arg Pro Ser Phe PheSer Ile Ser 100 105 110 ccg cag gac tgc cag ttt gct ttg ggg ggc cgg ggtcag aag cca gag 384 Pro Gln Asp Cys Gln Phe Ala Leu Gly Gly Arg Gly GlnLys Pro Glu 115 120 125 ctg cag atg ctc act gtg tcg gag gag ctg ccg tttgac aac gtg gat 432 Leu Gln Met Leu Thr Val Ser Glu Glu Leu Pro Phe AspAsn Val Asp 130 135 140 ggt ggt gtg tgg agg caa ggc ttc gac atc tcc tacgac ccg cac gac 480 Gly Gly Val Trp Arg Gln Gly Phe Asp Ile Ser Tyr AspPro His Asp 145 150 155 160 tgg gat gct gaa gac ctg cag gtg ttt gtg gtgccc cac tct cac aat 528 Trp Asp Ala Glu Asp Leu Gln Val Phe Val Val ProHis Ser His Asn 165 170 175 gac cca ggc tgg atc aag acc ttt gac aag tactac aca gag cag acc 576 Asp Pro Gly Trp Ile Lys Thr Phe Asp Lys Tyr TyrThr Glu Gln Thr 180 185 190 caa cac atc ctc aat agc atg gtg tct aag ctgcag gag gac ccc cgg 624 Gln His Ile Leu Asn Ser Met Val Ser Lys Leu GlnGlu Asp Pro Arg 195 200 205 cgg cgc ttc ctc tgg gca gag gtc tcc ttc ttcgcc aag tgg tgg gac 672 Arg Arg Phe Leu Trp Ala Glu Val Ser Phe Phe AlaLys Trp Trp Asp 210 215 220 aac atc aat gtc caa aag aga gcg gca gtc cgaagg ctg gtg gga aac 720 Asn Ile Asn Val Gln Lys Arg Ala Ala Val Arg ArgLeu Val Gly Asn 225 230 235 240 ggg cag ctg gag att gcg aca gga ggc tgggtg atg cca gat gag gcc 768 Gly Gln Leu Glu Ile Ala Thr Gly Gly Trp ValMet Pro Asp Glu Ala 245 250 255 aat tcc cac tac ttt gca ttg att gac cagctc atc gaa gga cac cag 816 Asn Ser His Tyr Phe Ala Leu Ile Asp Gln LeuIle Glu Gly His Gln 260 265 270 tgg ctg gag aga aat ctt ggt gca acc ccccgc tct ggc tgg gca gtg 864 Trp Leu Glu Arg Asn Leu Gly Ala Thr Pro ArgSer Gly Trp Ala Val 275 280 285 gac ccc ttt gga tac agc tcc acc atg ccttac ctg ctg cgc cgt gcc 912 Asp Pro Phe Gly Tyr Ser Ser Thr Met Pro TyrLeu Leu Arg Arg Ala 290 295 300 aac ctc acc agc atg ctg att cag aga gtgcac tat gcc atc aag aag 960 Asn Leu Thr Ser Met Leu Ile Gln Arg Val HisTyr Ala Ile Lys Lys 305 310 315 320 cac ttt gct gcc acc cac agc cta gagttc atg tgg agg cag aca tgg 1008 His Phe Ala Ala Thr His Ser Leu Glu PheMet Trp Arg Gln Thr Trp 325 330 335 gac tcg gac tcc agc aca gac atc ttctgt cac atg atg ccc ttc tac 1056 Asp Ser Asp Ser Ser Thr Asp Ile Phe CysHis Met Met Pro Phe Tyr 340 345 350 agc tat gac gtc ccc cat acc tgt ggccca gat ccc aag atc tgc tgc 1104 Ser Tyr Asp Val Pro His Thr Cys Gly ProAsp Pro Lys Ile Cys Cys 355 360 365 caa ttt gat ttc aaa cgc ctg cct ggtggg cgc atc aac tgc cct tgg 1152 Gln Phe Asp Phe Lys Arg Leu Pro Gly GlyArg Ile Asn Cys Pro Trp 370 375 380 aag gtg cca ccc cgg gcc atc aca gaggcc aac gtg gca gag agg gca 1200 Lys Val Pro Pro Arg Ala Ile Thr Glu AlaAsn Val Ala Glu Arg Ala 385 390 395 400 gcc ctg ctt ctg gac caa tac cggaag aag tcc cag ctg ttc cga agc 1248 Ala Leu Leu Leu Asp Gln Tyr Arg LysLys Ser Gln Leu Phe Arg Ser 405 410 415 aac gtc ctc ctg gtg cct ctt ggagat gac ttc cga tat gac aag ccc 1296 Asn Val Leu Leu Val Pro Leu Gly AspAsp Phe Arg Tyr Asp Lys Pro 420 425 430 cag gag tgg gat gcc cag ttc ttcaac tac caa cgg ctc ttt gac ttc 1344 Gln Glu Trp Asp Ala Gln Phe Phe AsnTyr Gln Arg Leu Phe Asp Phe 435 440 445 ttc aac agc agg cct aac ctc catgtg cag gcc cag ttt ggc act ctt 1392 Phe Asn Ser Arg Pro Asn Leu His ValGln Ala Gln Phe Gly Thr Leu 450 455 460 tct gac tat ttt gat gcc ctg tacaag agg aca ggg gtg gag cca ggg 1440 Ser Asp Tyr Phe Asp Ala Leu Tyr LysArg Thr Gly Val Glu Pro Gly 465 470 475 480 gcc cgg cct cca ggg ttt cctgtg ctg agc ggg gat ttc ttc tcc tat 1488 Ala Arg Pro Pro Gly Phe Pro ValLeu Ser Gly Asp Phe Phe Ser Tyr 485 490 495 gcg gac cgg gag gat cat tactgg aca ggc tat tac act tcc cgg ccc 1536 Ala Asp Arg Glu Asp His Tyr TrpThr Gly Tyr Tyr Thr Ser Arg Pro 500 505 510 ttc tac aag agc tta gac cgagtc ctg gaa gcc cac ctg cgg ggg gca 1584 Phe Tyr Lys Ser Leu Asp Arg ValLeu Glu Ala His Leu Arg Gly Ala 515 520 525 gag gtt ctg tac agc ctg gctgca gct cac gct cgc cgc tct ggt ctg 1632 Glu Val Leu Tyr Ser Leu Ala AlaAla His Ala Arg Arg Ser Gly Leu 530 535 540 gct ggc cgg tac cca ctg tctgat ttc acc ctc ctg acg gaa gct cgg 1680 Ala Gly Arg Tyr Pro Leu Ser AspPhe Thr Leu Leu Thr Glu Ala Arg 545 550 555 560 cgc aca ttg ggg ctc ttccag cat cac gat gcc atc act ggc acg gcc 1728 Arg Thr Leu Gly Leu Phe GlnHis His Asp Ala Ile Thr Gly Thr Ala 565 570 575 aag gag gct gtg gtg gtggac tat ggg gtc agg ctt ctg cgc tcc ctt 1776 Lys Glu Ala Val Val Val AspTyr Gly Val Arg Leu Leu Arg Ser Leu 580 585 590 gtc aac ctg aag cag gtcatc att cat gca gcc cac tat ctg gtg ctg 1824 Val Asn Leu Lys Gln Val IleIle His Ala Ala His Tyr Leu Val Leu 595 600 605 ggg gac aag gag acc taccac ttt gac cct gag gcg ccc ttc ctc caa 1872 Gly Asp Lys Glu Thr Tyr HisPhe Asp Pro Glu Ala Pro Phe Leu Gln 610 615 620 gtg gat gac act cgc ttaagt cac gac gcc ctc cca gag cgc acg gtg 1920 Val Asp Asp Thr Arg Leu SerHis Asp Ala Leu Pro Glu Arg Thr Val 625 630 635 640 atc cag ctg gat tcctcg ccc agg ttt gtg gtc cta ttc aac cca ctg 1968 Ile Gln Leu Asp Ser SerPro Arg Phe Val Val Leu Phe Asn Pro Leu 645 650 655 gaa cag gag cga ttcagc atg gtg tcc ctg ctg gtc aac tct ccc cgc 2016 Glu Gln Glu Arg Phe SerMet Val Ser Leu Leu Val Asn Ser Pro Arg 660 665 670 gtg cgt gtc ctt tcggag gag ggt cag ccc ctg gcc gtg cag atc agc 2064 Val Arg Val Leu Ser GluGlu Gly Gln Pro Leu Ala Val Gln Ile Ser 675 680 685 gca cac tgg agc tctgcc acc gag gcg gtc cct gac gtc tac cag gtg 2112 Ala His Trp Ser Ser AlaThr Glu Ala Val Pro Asp Val Tyr Gln Val 690 695 700 tct gtg cct gtc cgcctg cca gcc ctg ggc ctg ggc gtg ctg cag cta 2160 Ser Val Pro Val Arg LeuPro Ala Leu Gly Leu Gly Val Leu Gln Leu 705 710 715 720 cag ctg ggc ctggat ggg cac cgc acg ctg ccc tcc tct gtg cgc atc 2208 Gln Leu Gly Leu AspGly His Arg Thr Leu Pro Ser Ser Val Arg Ile 725 730 735 tac ctg cac ggccgg cag ctg tcc gtc agc agg cac gaa gcg ttt cct 2256 Tyr Leu His Gly ArgGln Leu Ser Val Ser Arg His Glu Ala Phe Pro 740 745 750 ctc cgt gtc attgac tct ggc acc agc gac ttc gcc ctc agc aac cgc 2304 Leu Arg Val Ile AspSer Gly Thr Ser Asp Phe Ala Leu Ser Asn Arg 755 760 765 tac atg cag gtctgg ttc tca ggc ctt act ggg ctc ctc aag agc atc 2352 Tyr Met Gln Val TrpPhe Ser Gly Leu Thr Gly Leu Leu Lys Ser Ile 770 775 780 cga agg gtg gatgag gag cac gag cag cag gtg gac atg cag gtc ctt 2400 Arg Arg Val Asp GluGlu His Glu Gln Gln Val Asp Met Gln Val Leu 785 790 795 800 gtc tat ggcacc cgt acg tcc aaa gac aag agt gga gcc tac ctc ttc 2448 Val Tyr Gly ThrArg Thr Ser Lys Asp Lys Ser Gly Ala Tyr Leu Phe 805 810 815 ctg ccc gatggc gag gct agc cct acg tcc cca agg agc ccc ccg tgc 2496 Leu Pro Asp GlyGlu Ala Ser Pro Thr Ser Pro Arg Ser Pro Pro Cys 820 825 830 tgc gtg tcactg aag gcc ctt tct tct cag agg tgg ttg cgt act atg 2544 Cys Val Ser LeuLys Ala Leu Ser Ser Gln Arg Trp Leu Arg Thr Met 835 840 845 agc aca ttcacc agg cgg tcc ggc ttt aca atc tgc cag ggg tgg agg 2592 Ser Thr Phe ThrArg Arg Ser Gly Phe Thr Ile Cys Gln Gly Trp Arg 850 855 860 ggc tgt ctctgg aca tat cat ccc tgg tgg aca tcc ggg act acg tca 2640 Gly Cys Leu TrpThr Tyr His Pro Trp Trp Thr Ser Gly Thr Thr Ser 865 870 875 880 aca aggagc tgg ccc tgc aca tcc ata cag aca tcg aca gcc agg gtg 2688 Thr Arg SerTrp Pro Cys Thr Ser Ile Gln Thr Ser Thr Ala Arg Val 885 890 895 cag ccccga cgg tat ctg aag aag ctc ccc ctc cag gcc aac ttc tac 2736 Gln Pro ArgArg Tyr Leu Lys Lys Leu Pro Leu Gln Ala Asn Phe Tyr 900 905 910 ccc atgcca gtc atg gcc tat atc cag gac gca cag aag cgc ctc acg 2784 Pro Met ProVal Met Ala Tyr Ile Gln Asp Ala Gln Lys Arg Leu Thr 915 920 925 ctg cacact gcc cag gcc ctg ggt gtc tct agc ctc aaa gat ggc cag 2832 Leu His ThrAla Gln Ala Leu Gly Val Ser Ser Leu Lys Asp Gly Gln 930 935 940 ctg gaggtg atc ttg gac cgg cgg ctg atg cag gat gac aac cgg ggc 2880 Leu Glu ValIle Leu Asp Arg Arg Leu Met Gln Asp Asp Asn Arg Gly 945 950 955 960 ctaggc caa ggg ctc aag gac aac aag aga acc tgc aac cgt ttc cgc 2928 Leu GlyGln Gly Leu Lys Asp Asn Lys Arg Thr Cys Asn Arg Phe Arg 965 970 975 ctcctg cta gag cgg cga acc gtg ggc agt gag gtc caa gat agc cac 2976 Leu LeuLeu Glu Arg Arg Thr Val Gly Ser Glu Val Gln Asp Ser His 980 985 990 tctacc agc tac cca tcc ctc ctc agc cac ctg acc tcc atg tac ctg 3024 Ser ThrSer Tyr Pro Ser Leu Leu Ser His Leu Thr Ser Met Tyr Leu 995 1000 1005aac gcc ccg gcg ctc gct ctg cct gta gcc agg atg cag ctc cca ggc 3072 AsnAla Pro Ala Leu Ala Leu Pro Val Ala Arg Met Gln Leu Pro Gly 1010 10151020 cct ggt ctg cgc tca ttt cat cct ctg gct tcc tca ctg ccc tgt gac3120 Pro Gly Leu Arg Ser Phe His Pro Leu Ala Ser Ser Leu Pro Cys Asp1025 1030 1035 1040 ttc cac ctg ctc aac cta cgt acg ctc cag gct gag gaggac acc cta 3168 Phe His Leu Leu Asn Leu Arg Thr Leu Gln Ala Glu Glu AspThr Leu 1045 1050 1055 ccc tcg gcg gag acc gca ctc atc tta cac cgc aagggt ttt gac tgc 3216 Pro Ser Ala Glu Thr Ala Leu Ile Leu His Arg Lys GlyPhe Asp Cys 1060 1065 1070 ggc ctg gag gcc aag aac ttg ggc ttc aac tgcacc aca agc caa ggc 3264 Gly Leu Glu Ala Lys Asn Leu Gly Phe Asn Cys ThrThr Ser Gln Gly 1075 1080 1085 aag gta gcc ctg ggc agc ctt ttc cat ggcctg gat gtg gta ttc ctt 3312 Lys Val Ala Leu Gly Ser Leu Phe His Gly LeuAsp Val Val Phe Leu 1090 1095 1100 cag cca acc tcc ttg acg tta ctg taccct ctg gcc tcc ccg tcc aac 3360 Gln Pro Thr Ser Leu Thr Leu Leu Tyr ProLeu Ala Ser Pro Ser Asn 1105 1110 1115 1120 agc act gac gtc tat ttg gagccc atg gag att gct acc ttt cgc ctc 3408 Ser Thr Asp Val Tyr Leu Glu ProMet Glu Ile Ala Thr Phe Arg Leu 1125 1130 1135 cgc ttg ggt tag 3420 ArgLeu Gly 57 3393 DNA Spodoptera frugiperda CDS (1)..(3390) 57 atg agg actcgt gtc ctt cgt tgc cgg ccg ttc tcc acc cgg atc ctg 48 Met Arg Thr ArgVal Leu Arg Cys Arg Pro Phe Ser Thr Arg Ile Leu 1 5 10 15 ctg ctg ctgcta ttt gtc ctt gcg ttt ggg gtc tac tgc tat ttc tac 96 Leu Leu Leu LeuPhe Val Leu Ala Phe Gly Val Tyr Cys Tyr Phe Tyr 20 25 30 aat gca tct cctcag aac tat aac aaa cca aga atc agt tac cca gcc 144 Asn Ala Ser Pro GlnAsn Tyr Asn Lys Pro Arg Ile Ser Tyr Pro Ala 35 40 45 agt atg gag cac ttcaaa tct tcc ctc act cac acc gtc aag agc cga 192 Ser Met Glu His Phe LysSer Ser Leu Thr His Thr Val Lys Ser Arg 50 55 60 gac gag cca act ccg gatcaa tgc cct gca ttg aag gaa agc gaa gcg 240 Asp Glu Pro Thr Pro Asp GlnCys Pro Ala Leu Lys Glu Ser Glu Ala 65 70 75 80 gac atc gac acc gtg gcgata tac cca act ttt gat ttt cag ccg agc 288 Asp Ile Asp Thr Val Ala IleTyr Pro Thr Phe Asp Phe Gln Pro Ser 85 90 95 tgg ttg cgt aca aag gaa ttttgg gac aag tcc ttc gag gat cgg tat 336 Trp Leu Arg Thr Lys Glu Phe TrpAsp Lys Ser Phe Glu Asp Arg Tyr 100 105 110 gaa aga att cat aac gac actaca cgg cct aga ctg aag gta atc gtg 384 Glu Arg Ile His Asn Asp Thr ThrArg Pro Arg Leu Lys Val Ile Val 115 120 125 gtt cct cac tca cac aac gacccg gga tgg ctg aag acg ttt gaa cag 432 Val Pro His Ser His Asn Asp ProGly Trp Leu Lys Thr Phe Glu Gln 130 135 140 tac ttc gag tgg aag acc aagaac att atc aac aac ata gtg aac aaa 480 Tyr Phe Glu Trp Lys Thr Lys AsnIle Ile Asn Asn Ile Val Asn Lys 145 150 155 160 ctg cac cag tac ccc aacatg acc ttc att tgg acc gag ata tcg ttt 528 Leu His Gln Tyr Pro Asn MetThr Phe Ile Trp Thr Glu Ile Ser Phe 165 170 175 ctg aat gcc tgg tgg gaaagg tcg cac cct gtc aaa caa aag gca ttg 576 Leu Asn Ala Trp Trp Glu ArgSer His Pro Val Lys Gln Lys Ala Leu 180 185 190 aaa aaa ctt atc aaa gaaggt cgt ctc gag atc acg acg ggc ggc tgg 624 Lys Lys Leu Ile Lys Glu GlyArg Leu Glu Ile Thr Thr Gly Gly Trp 195 200 205 gtg atg ccg gac gaa gcctgc acg cat atc tat gcg cta att gac cag 672 Val Met Pro Asp Glu Ala CysThr His Ile Tyr Ala Leu Ile Asp Gln 210 215 220 ttt att gaa gga cat cactgg gtg aaa act aat ctc ggc gtc atc ccg 720 Phe Ile Glu Gly His His TrpVal Lys Thr Asn Leu Gly Val Ile Pro 225 230 235 240 aag aca gga tgg tctatt gac ccc ttc ggc cac ggg gcc act gtg cct 768 Lys Thr Gly Trp Ser IleAsp Pro Phe Gly His Gly Ala Thr Val Pro 245 250 255 tac ctg cta gac cagagc ggc ctt gag gga acc att ata cag aga atc 816 Tyr Leu Leu Asp Gln SerGly Leu Glu Gly Thr Ile Ile Gln Arg Ile 260 265 270 cat tat gcg tgg aaacag tgg ctg gcg gag cga cag att gag gag ttt 864 His Tyr Ala Trp Lys GlnTrp Leu Ala Glu Arg Gln Ile Glu Glu Phe 275 280 285 tac tgg ctg gcg agttgg gct act acg aag ccg tcc atg ata gtg cac 912 Tyr Trp Leu Ala Ser TrpAla Thr Thr Lys Pro Ser Met Ile Val His 290 295 300 aat cag ccg ttt gatatt tat tca ata aaa agc acg tgt ggc ccg cac 960 Asn Gln Pro Phe Asp IleTyr Ser Ile Lys Ser Thr Cys Gly Pro His 305 310 315 320 cct tca att tgtctc agt ttc gac ttc agg aag att ccc ggc gaa tat 1008 Pro Ser Ile Cys LeuSer Phe Asp Phe Arg Lys Ile Pro Gly Glu Tyr 325 330 335 tct gaa tac acagct aag cac gaa gac atc acg gaa cac aac ttg cac 1056 Ser Glu Tyr Thr AlaLys His Glu Asp Ile Thr Glu His Asn Leu His 340 345 350 agc aag gca aagact ttg ata gag gag tac gac cgt atc ggg tcc ctg 1104 Ser Lys Ala Lys ThrLeu Ile Glu Glu Tyr Asp Arg Ile Gly Ser Leu 355 360 365 act cca cac aacgtg gtg ctg gtg ccg ctc gga gac gac ttc aga tac 1152 Thr Pro His Asn ValVal Leu Val Pro Leu Gly Asp Asp Phe Arg Tyr 370 375 380 gag tac agc gtcgag ttt gat gcc caa tac gtc aat tat atg aaa atg 1200 Glu Tyr Ser Val GluPhe Asp Ala Gln Tyr Val Asn Tyr Met Lys Met 385 390 395 400 ttt aac tacatc aat gct cac aag gaa atc ttc aac gct gac gta cag 1248 Phe Asn Tyr IleAsn Ala His Lys Glu Ile Phe Asn Ala Asp Val Gln 405 410 415 ttc gga actcct ctc gat tac ttt aac gcc atg aaa gaa aga cat caa 1296 Phe Gly Thr ProLeu Asp Tyr Phe Asn Ala Met Lys Glu Arg His Gln 420 425 430 aat ata cccagc tta aag gga gat ttc ttc gtt tac tcc gat att ttc 1344 Asn Ile Pro SerLeu Lys Gly Asp Phe Phe Val Tyr Ser Asp Ile Phe 435 440 445 agc gaa ggtaaa cca gcg tac tgg tca ggt tac tac act act aga ccc 1392 Ser Glu Gly LysPro Ala Tyr Trp Ser Gly Tyr Tyr Thr Thr Arg Pro 450 455 460 tac caa aaaatc ctc gcc cgt cag ttc gaa cac caa ctg cga tcg gca 1440 Tyr Gln Lys IleLeu Ala Arg Gln Phe Glu His Gln Leu Arg Ser Ala 465 470 475 480 gag atttta ttc acc ctt gta tcg aac tac atc aga cag atg ggt cgc 1488 Glu Ile LeuPhe Thr Leu Val Ser Asn Tyr Ile Arg Gln Met Gly Arg 485 490 495 caa ggagag ttc gga gct tct gag aaa aag tta gaa aaa tct tac gag 1536 Gln Gly GluPhe Gly Ala Ser Glu Lys Lys Leu Glu Lys Ser Tyr Glu 500 505 510 cag cttatc tat gct cga cgg aac ttg ggt ctg ttt caa cat cac gat 1584 Gln Leu IleTyr Ala Arg Arg Asn Leu Gly Leu Phe Gln His His Asp 515 520 525 gcg attact gga aca tca aag tcc agt gtg atg caa gat tac gga acc 1632 Ala Ile ThrGly Thr Ser Lys Ser Ser Val Met Gln Asp Tyr Gly Thr 530 535 540 aaa ctgttc aca agt ctg tat cac tgc atc cgc ctg cag gag gcc gcg 1680 Lys Leu PheThr Ser Leu Tyr His Cys Ile Arg Leu Gln Glu Ala Ala 545 550 555 560 ctcacc acc atc atg ttg cct gac cag tcg ttg cac tcg cag agc att 1728 Leu ThrThr Ile Met Leu Pro Asp Gln Ser Leu His Ser Gln Ser Ile 565 570 575 atacaa agc gag gtt gag tgg gaa act tac gga aaa ccg ccc aag aag 1776 Ile GlnSer Glu Val Glu Trp Glu Thr Tyr Gly Lys Pro Pro Lys Lys 580 585 590 ctgcaa gtg tcc ttc att gac aag aag aaa gtt ata ctt ttt aat ccg 1824 Leu GlnVal Ser Phe Ile Asp Lys Lys Lys Val Ile Leu Phe Asn Pro 595 600 605 ttggct gag act cga act gaa gtg gtc acg gtt aga tcc aac acg tcc 1872 Leu AlaGlu Thr Arg Thr Glu Val Val Thr Val Arg Ser Asn Thr Ser 610 615 620 aacatc cgg gtg tac gat aca cac aag agg aag cac gtc ttg tat cag 1920 Asn IleArg Val Tyr Asp Thr His Lys Arg Lys His Val Leu Tyr Gln 625 630 635 640ata atg ccc agc atc aca atc caa gac aac ggc aag agt atc gta agc 1968 IleMet Pro Ser Ile Thr Ile Gln Asp Asn Gly Lys Ser Ile Val Ser 645 650 655gac acc acg ttc gac ata atg ttc gtg gcc acc atc ccg ccc ctc acc 2016 AspThr Thr Phe Asp Ile Met Phe Val Ala Thr Ile Pro Pro Leu Thr 660 665 670tcc atc tcg tac aag ctg cag gag cac acc aac act tcc cac cac tgc 2064 SerIle Ser Tyr Lys Leu Gln Glu His Thr Asn Thr Ser His His Cys 675 680 685gtc att ttc tgc aac aac tgc gaa caa tac cag aaa tcc aat gtg ttc 2112 ValIle Phe Cys Asn Asn Cys Glu Gln Tyr Gln Lys Ser Asn Val Phe 690 695 700caa att aag aaa atg atg cct ggt gac ata caa tta gaa aat gca gtg 2160 GlnIle Lys Lys Met Met Pro Gly Asp Ile Gln Leu Glu Asn Ala Val 705 710 715720 cta aaa ctt ctc gtt aat agg aac acc ggc ttt ctg aga caa gtc tat 2208Leu Lys Leu Leu Val Asn Arg Asn Thr Gly Phe Leu Arg Gln Val Tyr 725 730735 aga aag gac atc cgg aag aga act gtc gtt gac gta caa ttc ggc gca 2256Arg Lys Asp Ile Arg Lys Arg Thr Val Val Asp Val Gln Phe Gly Ala 740 745750 tat caa agt gcc caa aga cat tct ggt gct tac ctc ttc atg cct cat 2304Tyr Gln Ser Ala Gln Arg His Ser Gly Ala Tyr Leu Phe Met Pro His 755 760765 tac gac tca cct gag aag aat gtt ctg cat ccc tac act aat cag aac 2352Tyr Asp Ser Pro Glu Lys Asn Val Leu His Pro Tyr Thr Asn Gln Asn 770 775780 aac atg caa gat gat aac ata atc ata gtg tcc gga cct att tct acg 2400Asn Met Gln Asp Asp Asn Ile Ile Ile Val Ser Gly Pro Ile Ser Thr 785 790795 800 gaa atc acg acc atg tac ttg ccc ttc ttg gtg cac act att agg ata2448 Glu Ile Thr Thr Met Tyr Leu Pro Phe Leu Val His Thr Ile Arg Ile 805810 815 tac aac gtg ccg gac ccg gta ctg tcg cgt gct att cta tta gag acc2496 Tyr Asn Val Pro Asp Pro Val Leu Ser Arg Ala Ile Leu Leu Glu Thr 820825 830 gat gta gat ttc gag gcg cca cct aag aac aga gag act gag tta ttt2544 Asp Val Asp Phe Glu Ala Pro Pro Lys Asn Arg Glu Thr Glu Leu Phe 835840 845 atg aga tta cag act gat ata caa aac ggt gac att ccc gaa ttt tac2592 Met Arg Leu Gln Thr Asp Ile Gln Asn Gly Asp Ile Pro Glu Phe Tyr 850855 860 acc gat cag aac gga ttc cag tac caa aag agg gtc aaa gtg aat aaa2640 Thr Asp Gln Asn Gly Phe Gln Tyr Gln Lys Arg Val Lys Val Asn Lys 865870 875 880 cta gga ata gaa gct aat tac tac ccg atc act acc atg gcg tgcctg 2688 Leu Gly Ile Glu Ala Asn Tyr Tyr Pro Ile Thr Thr Met Ala Cys Leu885 890 895 caa gac gag gag acc cgg ctc act ctg ctg acg aac cac gct caaggc 2736 Gln Asp Glu Glu Thr Arg Leu Thr Leu Leu Thr Asn His Ala Gln Gly900 905 910 gct gct gca tac gaa cca gga cgc tta gaa gtc atg ctc gat cgtcga 2784 Ala Ala Ala Tyr Glu Pro Gly Arg Leu Glu Val Met Leu Asp Arg Arg915 920 925 act ctt tat gat gac ttc aga gga atc ggt gaa gga gta gtc gataac 2832 Thr Leu Tyr Asp Asp Phe Arg Gly Ile Gly Glu Gly Val Val Asp Asn930 935 940 aaa ccg acg act ttc cag aac tgg att tta att gaa tcc atg ccaggc 2880 Lys Pro Thr Thr Phe Gln Asn Trp Ile Leu Ile Glu Ser Met Pro Gly945 950 955 960 gtg acg cga gcc aag aga gac act agt gaa cca ggt ttc aaattt gtt 2928 Val Thr Arg Ala Lys Arg Asp Thr Ser Glu Pro Gly Phe Lys PheVal 965 970 975 aat gaa cgt cgt ttt ggc ccc ggc cag aag gaa agc cct taccaa gta 2976 Asn Glu Arg Arg Phe Gly Pro Gly Gln Lys Glu Ser Pro Tyr GlnVal 980 985 990 ccg tcg cag act gcg gac tac ctg agc agg atg ttc aat tacccg gtg 3024 Pro Ser Gln Thr Ala Asp Tyr Leu Ser Arg Met Phe Asn Tyr ProVal 995 1000 1005 aac gtg tac ctg gtg gac act agc gag gtt ggc gag atcgag gtg aag 3072 Asn Val Tyr Leu Val Asp Thr Ser Glu Val Gly Glu Ile GluVal Lys 1010 1015 1020 ccg tac cag tcg ttc ctg cag agc ttc ccg ccc ggcatc cac ctg gtc 3120 Pro Tyr Gln Ser Phe Leu Gln Ser Phe Pro Pro Gly IleHis Leu Val 1025 1030 1035 1040 acc ctg cgc acc atc acc gac gac gtg ctcgaa ctc ttc ccc agc aac 3168 Thr Leu Arg Thr Ile Thr Asp Asp Val Leu GluLeu Phe Pro Ser Asn 1045 1050 1055 gaa agc tac atg gta ctg cac cga ccagga tac agc tgc gct gtc gga 3216 Glu Ser Tyr Met Val Leu His Arg Pro GlyTyr Ser Cys Ala Val Gly 1060 1065 1070 gag aag cca gtc gcc aag tct cccaag ttt tcg tcc aaa acc agg ttc 3264 Glu Lys Pro Val Ala Lys Ser Pro LysPhe Ser Ser Lys Thr Arg Phe 1075 1080 1085 aat ggt ctg aac att cag aacatc act gca gtc agc ctg acc ggc ctg 3312 Asn Gly Leu Asn Ile Gln Asn IleThr Ala Val Ser Leu Thr Gly Leu 1090 1095 1100 aag tca ctc cga cct ctcaca ggt ctg agt gac atc cac ctg aac gct 3360 Lys Ser Leu Arg Pro Leu ThrGly Leu Ser Asp Ile His Leu Asn Ala 1105 1110 1115 1120 atg gag gta aaaact tac aag atc agg ttt taa 3393 Met Glu Val Lys Thr Tyr Lys Ile Arg Phe1125 1130 58 3033 DNA Homo sapiens CDS (1)..(3030) 58 atg ggc tac gcgcgg gct tcg ggg gtc tgc gct cgc ggc tgc ctg gac 48 Met Gly Tyr Ala ArgAla Ser Gly Val Cys Ala Arg Gly Cys Leu Asp 1 5 10 15 tca gca ggc ccctgg acc atg tcc cgc gcc ctg cgg cca ccg ctc ccg 96 Ser Ala Gly Pro TrpThr Met Ser Arg Ala Leu Arg Pro Pro Leu Pro 20 25 30 cct ctc tgc ttt ttcctt ttg ttg ctg gcg gct gcc ggt gct cgg gcc 144 Pro Leu Cys Phe Phe LeuLeu Leu Leu Ala Ala Ala Gly Ala Arg Ala 35 40 45 ggg gga tac gag aca tgcccc aca gtg cag ccg aac atg ctg aac gtg 192 Gly Gly Tyr Glu Thr Cys ProThr Val Gln Pro Asn Met Leu Asn Val 50 55 60 cac ctg ctg cct cac aca catgat gac gtg ggc tgg ctc aaa acc gtg 240 His Leu Leu Pro His Thr His AspAsp Val Gly Trp Leu Lys Thr Val 65 70 75 80 gac cag tac ttt tat gga atcaag aat gac atc cag cac gcc ggt gtg 288 Asp Gln Tyr Phe Tyr Gly Ile LysAsn Asp Ile Gln His Ala Gly Val 85 90 95 cag tac atc ctg gac tcg gtc atctct gcc ttg ctg gca gat ccc acc 336 Gln Tyr Ile Leu Asp Ser Val Ile SerAla Leu Leu Ala Asp Pro Thr 100 105 110 cgt cgc ttc att tac gtg gag attgcc ttc ttc tcc cgt tgg tgg cac 384 Arg Arg Phe Ile Tyr Val Glu Ile AlaPhe Phe Ser Arg Trp Trp His 115 120 125 cag cag aca aat gcc aca cag gaagtc gtg cga gac ctt gtg cgc cag 432 Gln Gln Thr Asn Ala Thr Gln Glu ValVal Arg Asp Leu Val Arg Gln 130 135 140 ggg cgc ctg gag ttc gcc aat ggtggc tgg gtg atg aac gat gag gca 480 Gly Arg Leu Glu Phe Ala Asn Gly GlyTrp Val Met Asn Asp Glu Ala 145 150 155 160 gcc acc cac tac ggt gcc atcgtg gac cag atg aca ctt ggg ctg cgc 528 Ala Thr His Tyr Gly Ala Ile ValAsp Gln Met Thr Leu Gly Leu Arg 165 170 175 ttt ctg gag gac aca ttt ggcaat gat ggg cga ccc cgt gtg gcc tgg 576 Phe Leu Glu Asp Thr Phe Gly AsnAsp Gly Arg Pro Arg Val Ala Trp 180 185 190 cac att gac ccc ttc ggc cactct cgg gag cag gcc tcg ctg ttt gcg 624 His Ile Asp Pro Phe Gly His SerArg Glu Gln Ala Ser Leu Phe Ala 195 200 205 cag atg ggc ttc gac ggc ttcttc ttt ggg cgc ctt gat tat caa gat 672 Gln Met Gly Phe Asp Gly Phe PhePhe Gly Arg Leu Asp Tyr Gln Asp 210 215 220 aag tgg gta cgg atg cag aagctg gag atg gag cag gtg tgg cgg gcc 720 Lys Trp Val Arg Met Gln Lys LeuGlu Met Glu Gln Val Trp Arg Ala 225 230 235 240 agc acc agc ctg aag cccccg acc gcg gac ctc ttc act ggt gtg ctt 768 Ser Thr Ser Leu Lys Pro ProThr Ala Asp Leu Phe Thr Gly Val Leu 245 250 255 ccc aat ggt tac aac ccgcca agg aat ctg tgc tgg gat gtg ctg tgt 816 Pro Asn Gly Tyr Asn Pro ProArg Asn Leu Cys Trp Asp Val Leu Cys 260 265 270 gtc gat cag ccg ctg gtggag gac cct cgc agc ccc gag tac aac gcc 864 Val Asp Gln Pro Leu Val GluAsp Pro Arg Ser Pro Glu Tyr Asn Ala 275 280 285 aag gag ctg gtc gat tacttc cta aat gtg gcc act gcc cag ggc cgg 912 Lys Glu Leu Val Asp Tyr PheLeu Asn Val Ala Thr Ala Gln Gly Arg 290 295 300 tat tac cgc acc aac cacact gtg atg acc atg ggc tcg gac ttc caa 960 Tyr Tyr Arg Thr Asn His ThrVal Met Thr Met Gly Ser Asp Phe Gln 305 310 315 320 tat gag aat gcc aacatg tgg ttc aag aac ctt gac aag ctc atc cgg 1008 Tyr Glu Asn Ala Asn MetTrp Phe Lys Asn Leu Asp Lys Leu Ile Arg 325 330 335 ctg gta aat gcg cagcag gca aaa gga agc agt gtc cat gtt ctc tac 1056 Leu Val Asn Ala Gln GlnAla Lys Gly Ser Ser Val His Val Leu Tyr 340 345 350 tcc acc ccc gct tgttac ctc tgg gag ctg aac aag gcc aac ctc acc 1104 Ser Thr Pro Ala Cys TyrLeu Trp Glu Leu Asn Lys Ala Asn Leu Thr 355 360 365 tgg tca gtg aaa catgac gac ttc ttc cct tac gcg gat ggc ccc cac 1152 Trp Ser Val Lys His AspAsp Phe Phe Pro Tyr Ala Asp Gly Pro His 370 375 380 cag ttc tgg acc ggttac ttt tcc agt cgg ccg gcc ctc aaa cgc tac 1200 Gln Phe Trp Thr Gly TyrPhe Ser Ser Arg Pro Ala Leu Lys Arg Tyr 385 390 395 400 gag cgc ctc agctac aac ttc ctg cag gtg tgc aac cag ctg gag gcg 1248 Glu Arg Leu Ser TyrAsn Phe Leu Gln Val Cys Asn Gln Leu Glu Ala 405 410 415 ctg gtg ggc ctggcg gcc aac gtg gga ccc tat ggc tcc gga gac agt 1296 Leu Val Gly Leu AlaAla Asn Val Gly Pro Tyr Gly Ser Gly Asp Ser 420 425 430 gca ccc ctc aatgag gcg atg gct gtg ctc cag cat cac gac gcc gtc 1344 Ala Pro Leu Asn GluAla Met Ala Val Leu Gln His His Asp Ala Val 435 440 445 agc ggc acc tcccgc cag cac gtg gcc aac gac tac gcg cgc cag ctt 1392 Ser Gly Thr Ser ArgGln His Val Ala Asn Asp Tyr Ala Arg Gln Leu 450 455 460 gcg gca ggc tggggg cct tgc gag gtt ctt ctg agc aac gcg ctg gcg 1440 Ala Ala Gly Trp GlyPro Cys Glu Val Leu Leu Ser Asn Ala Leu Ala 465 470 475 480 cgg ctc agaggc ttc aaa gat cac ttc acc ttt tgc caa cag cta aac 1488 Arg Leu Arg GlyPhe Lys Asp His Phe Thr Phe Cys Gln Gln Leu Asn 485 490 495 atc agc atctgc ccg ctc agc cag acg gcg gcg cgc ttc cag gtc atc 1536 Ile Ser Ile CysPro Leu Ser Gln Thr Ala Ala Arg Phe Gln Val Ile 500 505 510 gtt tat aatccc ctg ggg cgg aag gtg aat tgg atg gta cgg ctg ccg 1584 Val Tyr Asn ProLeu Gly Arg Lys Val Asn Trp Met Val Arg Leu Pro 515 520 525 gtc agc gaaggc gtt ttc gtt gtg aag gac ccc aat ggc agg aca gtg 1632 Val Ser Glu GlyVal Phe Val Val Lys Asp Pro Asn Gly Arg Thr Val 530 535 540 ccc agc gatgtg gta ata ttt ccc agc tca gac agc cag gcg cac cct 1680 Pro Ser Asp ValVal Ile Phe Pro Ser Ser Asp Ser Gln Ala His Pro 545 550 555 560 ccg gagctg ctg ttc tca gcc tca ctg ccc gcc ctg ggc ttc agc acc 1728 Pro Glu LeuLeu Phe Ser Ala Ser Leu Pro Ala Leu Gly Phe Ser Thr 565 570 575 tat tcagta gcc cag gtg cct cgc tgg aag ccc cag gcc cgc gca cca 1776 Tyr Ser ValAla Gln Val Pro Arg Trp Lys Pro Gln Ala Arg Ala Pro 580 585 590 cag cccatc ccc aga aga tcc tgg tcc cct gct tta acc atc gaa aat 1824 Gln Pro IlePro Arg Arg Ser Trp Ser Pro Ala Leu Thr Ile Glu Asn 595 600 605 gag cacatc cgg gca acg ttt gat cct gac aca ggg ctg ttg atg gag 1872 Glu His IleArg Ala Thr Phe Asp Pro Asp Thr Gly Leu Leu Met Glu 610 615 620 att atgaac atg aat cag caa ctc ctg ctg cct gtt cgc cag acc ttc 1920 Ile Met AsnMet Asn Gln Gln Leu Leu Leu Pro Val Arg Gln Thr Phe 625 630 635 640 ttctgg tac aac gcc agt ata ggt gac aac gaa agt gac cag gcc tca 1968 Phe TrpTyr Asn Ala Ser Ile Gly Asp Asn Glu Ser Asp Gln Ala Ser 645 650 655 ggtgcc tac atc ttc aga ccc aac caa cag aaa ccg ctg cct gtg agc 2016 Gly AlaTyr Ile Phe Arg Pro Asn Gln Gln Lys Pro Leu Pro Val Ser 660 665 670 cgctgg gct cag atc cac ctg gtg aag aca ccc ttg gtg cag gag gtg 2064 Arg TrpAla Gln Ile His Leu Val Lys Thr Pro Leu Val Gln Glu Val 675 680 685 caccag aac ttc tca gct tgg tgt tcc cag gtg gtt cgc ctg tac cca 2112 His GlnAsn Phe Ser Ala Trp Cys Ser Gln Val Val Arg Leu Tyr Pro 690 695 700 ggacag cgg cac ctg gag cta gag tgg tcg gtg ggg ccg ata cct gtg 2160 Gly GlnArg His Leu Glu Leu Glu Trp Ser Val Gly Pro Ile Pro Val 705 710 715 720ggc gac acc tgg ggg aag gag gtc atc agc cgt ttt gac aca ccg ctg 2208 GlyAsp Thr Trp Gly Lys Glu Val Ile Ser Arg Phe Asp Thr Pro Leu 725 730 735gag aca aag gga cgc ttc tac aca gac agc aat ggc cgg gag atc ctg 2256 GluThr Lys Gly Arg Phe Tyr Thr Asp Ser Asn Gly Arg Glu Ile Leu 740 745 750gag agg agg cgg gat tat cga ccc acc tgg aaa ctg aac cag acg gag 2304 GluArg Arg Arg Asp Tyr Arg Pro Thr Trp Lys Leu Asn Gln Thr Glu 755 760 765ccc gtg gca gga aac tac tat cca gtc aac acc cgg att tac atc acg 2352 ProVal Ala Gly Asn Tyr Tyr Pro Val Asn Thr Arg Ile Tyr Ile Thr 770 775 780gat gga aac atg cag ctg act gtg ctg act gac cgc tcc cag ggg ggc 2400 AspGly Asn Met Gln Leu Thr Val Leu Thr Asp Arg Ser Gln Gly Gly 785 790 795800 agc agc ctg aga gat ggc tcg ctg gag ctc atg gtg cac cga agg ctg 2448Ser Ser Leu Arg Asp Gly Ser Leu Glu Leu Met Val His Arg Arg Leu 805 810815 ctg aag gac gat gga cgc gga gta tcg gag cca cta atg gag aac ggg 2496Leu Lys Asp Asp Gly Arg Gly Val Ser Glu Pro Leu Met Glu Asn Gly 820 825830 tcg ggg gcg tgg gtg cga ggg cgc cac ctg gtg ctg ctg gac aca gcc 2544Ser Gly Ala Trp Val Arg Gly Arg His Leu Val Leu Leu Asp Thr Ala 835 840845 cag gct gca gcc gcc gga cac cgg ctc ctg gcg gag cag gag gtc ctg 2592Gln Ala Ala Ala Ala Gly His Arg Leu Leu Ala Glu Gln Glu Val Leu 850 855860 gcc cct cag gtg gtg ctg gcc ccg ggt ggc ggc gcc gcc tac aat ctc 2640Ala Pro Gln Val Val Leu Ala Pro Gly Gly Gly Ala Ala Tyr Asn Leu 865 870875 880 ggg gct cct ccg cgc acg cag ttc tca ggg ctg cgc agg gac ctg ccg2688 Gly Ala Pro Pro Arg Thr Gln Phe Ser Gly Leu Arg Arg Asp Leu Pro 885890 895 ccc tcg gtg cac ctg ctc acg ctg gcc agc tgg ggc ccc gaa atg gtg2736 Pro Ser Val His Leu Leu Thr Leu Ala Ser Trp Gly Pro Glu Met Val 900905 910 ctg ctg cgc ttg gag cac cag ttt gcc gta gga gag gat tcc gga cgt2784 Leu Leu Arg Leu Glu His Gln Phe Ala Val Gly Glu Asp Ser Gly Arg 915920 925 aac ctg agc gcc ccc gtt acc ttg aac ttg agg gac ctg ttc tcc acc2832 Asn Leu Ser Ala Pro Val Thr Leu Asn Leu Arg Asp Leu Phe Ser Thr 930935 940 ttc acc atc acc cgc ctg cag gag acc acg ctg gtg gcc aac cag ctc2880 Phe Thr Ile Thr Arg Leu Gln Glu Thr Thr Leu Val Ala Asn Gln Leu 945950 955 960 cgc gag gca gcc tcc agg ctc aag tgg aca aca aac aca ggc cccaca 2928 Arg Glu Ala Ala Ser Arg Leu Lys Trp Thr Thr Asn Thr Gly Pro Thr965 970 975 ccc cac caa act ccg tac cag ctg gac ccg gcc aac atc acg ctggaa 2976 Pro His Gln Thr Pro Tyr Gln Leu Asp Pro Ala Asn Ile Thr Leu Glu980 985 990 ccc atg gaa atc cgc act ttc ctg gcc tca gtt caa tgg aag gaggtg 3024 Pro Met Glu Ile Arg Thr Phe Leu Ala Ser Val Gln Trp Lys Glu Val995 1000 1005 gat ggt tag 3033 Asp Gly 1010 59 3189 DNA Homo sapiens CDS(1)..(3186) 59 atg gcg gca gcg ccg ttc ttg aag cac tgg cgc acc act tttgag cgg 48 Met Ala Ala Ala Pro Phe Leu Lys His Trp Arg Thr Thr Phe GluArg 1 5 10 15 gtg gag aag ttc gtg tcc ccg atc tac ttc acc gac tgt aacctc cgc 96 Val Glu Lys Phe Val Ser Pro Ile Tyr Phe Thr Asp Cys Asn LeuArg 20 25 30 ggc agg ctt ttt ggg gcc agc tgc cct gtg gct gtg ctc tcc agcttc 144 Gly Arg Leu Phe Gly Ala Ser Cys Pro Val Ala Val Leu Ser Ser Phe35 40 45 ctg acg ccg gag aga ctt ccc tac cag gag gca gtc cag cgg gac ttc192 Leu Thr Pro Glu Arg Leu Pro Tyr Gln Glu Ala Val Gln Arg Asp Phe 5055 60 cgc ccc gcg cag gtc ggc gac agc ttc gga ccc aca tgg tgg acc tgc240 Arg Pro Ala Gln Val Gly Asp Ser Phe Gly Pro Thr Trp Trp Thr Cys 6570 75 80 tgg ttc cgg gtg gag ctg acc atc cca gag gca tgg gtg ggc cag gaa288 Trp Phe Arg Val Glu Leu Thr Ile Pro Glu Ala Trp Val Gly Gln Glu 8590 95 gtt cac ctt tgc tgg gaa agt gat gga gaa ggt ctg gtg tgg cgt gat336 Val His Leu Cys Trp Glu Ser Asp Gly Glu Gly Leu Val Trp Arg Asp 100105 110 gga gaa cct gtc cag ggt tta acc aaa gag ggt gag aag acc agc tat384 Gly Glu Pro Val Gln Gly Leu Thr Lys Glu Gly Glu Lys Thr Ser Tyr 115120 125 gtc ctg act gac agg ctg ggg gaa aga gac ccc cga agc ctc act ctc432 Val Leu Thr Asp Arg Leu Gly Glu Arg Asp Pro Arg Ser Leu Thr Leu 130135 140 tat gtg gaa gta gcc tgc aat ggg ctc ctg ggg gcc ggg aag gga agc480 Tyr Val Glu Val Ala Cys Asn Gly Leu Leu Gly Ala Gly Lys Gly Ser 145150 155 160 atg att gca gcc cct gac cct gag aag ata ttc cag ctg agc cgggct 528 Met Ile Ala Ala Pro Asp Pro Glu Lys Ile Phe Gln Leu Ser Arg Ala165 170 175 gag cta gct gtg ttc cac cgg gat gtc cac atg ctc ctg gtg gatctg 576 Glu Leu Ala Val Phe His Arg Asp Val His Met Leu Leu Val Asp Leu180 185 190 gag ctg ctg ctg ggc ata gcc aag ggc ctc ggg aag gac aac cagcgc 624 Glu Leu Leu Leu Gly Ile Ala Lys Gly Leu Gly Lys Asp Asn Gln Arg195 200 205 agc ttc cag gcc ctg tac aca gcc aat cag atg gtg aac gtg tgtgac 672 Ser Phe Gln Ala Leu Tyr Thr Ala Asn Gln Met Val Asn Val Cys Asp210 215 220 cct gcc cag ccc gag acc ttc cca gtg gcc cag gcc ctg gcc tccagg 720 Pro Ala Gln Pro Glu Thr Phe Pro Val Ala Gln Ala Leu Ala Ser Arg225 230 235 240 ttc ttt ggc caa cat ggg ggt gaa agc caa cac acc att catgcc aca 768 Phe Phe Gly Gln His Gly Gly Glu Ser Gln His Thr Ile His AlaThr 245 250 255 ggg cac tgc cac att gat aca gcc tgg ctt tgg ccc ttc aaagag act 816 Gly His Cys His Ile Asp Thr Ala Trp Leu Trp Pro Phe Lys GluThr 260 265 270 gtg agg aaa tgt gcc cgg agc tgg gtg acc gcc ctg cag ctcatg gag 864 Val Arg Lys Cys Ala Arg Ser Trp Val Thr Ala Leu Gln Leu MetGlu 275 280 285 cgg aac cct gag ttc atc ttt gcc tgc tcc cag gcg cag cagctg gaa 912 Arg Asn Pro Glu Phe Ile Phe Ala Cys Ser Gln Ala Gln Gln LeuGlu 290 295 300 tgg gtg aag agc cgc tac cct ggc ctg tac tcc cgc atc caggag ttt 960 Trp Val Lys Ser Arg Tyr Pro Gly Leu Tyr Ser Arg Ile Gln GluPhe 305 310 315 320 gcg tgc cgt ggg cag ttt gtg cct gtg ggg ggc acc tgggtg gaa atg 1008 Ala Cys Arg Gly Gln Phe Val Pro Val Gly Gly Thr Trp ValGlu Met 325 330 335 gat ggg aac ctg ccc agt gga gag gcc atg gtg agg cagttt ttg cag 1056 Asp Gly Asn Leu Pro Ser Gly Glu Ala Met Val Arg Gln PheLeu Gln 340 345 350 ggc cag aac ttc ttt ctg cag gag ttt ggg aag atg tgctct gag ttc 1104 Gly Gln Asn Phe Phe Leu Gln Glu Phe Gly Lys Met Cys SerGlu Phe 355 360 365 tgg ctg ccg gac acc ttt ggc tac tca gca cag ctc ccccag atc atg 1152 Trp Leu Pro Asp Thr Phe Gly Tyr Ser Ala Gln Leu Pro GlnIle Met 370 375 380 cac ggc tgt ggc atc agg cgc ttt ctc acc cag aaa ttgagc tgg aat 1200 His Gly Cys Gly Ile Arg Arg Phe Leu Thr Gln Lys Leu SerTrp Asn 385 390 395 400 ttg gtg aac tcc ttc cca cac cat aca ttt ttc tgggag ggc ctg gat 1248 Leu Val Asn Ser Phe Pro His His Thr Phe Phe Trp GluGly Leu Asp 405 410 415 ggc tcc cgt gta ctg gtc cac ttc cca cct ggc gactcc tat ggg atg 1296 Gly Ser Arg Val Leu Val His Phe Pro Pro Gly Asp SerTyr Gly Met 420 425 430 cag ggc agc gtg gag gag gtg ctg aag acc gtg gccaac aac cgg gac 1344 Gln Gly Ser Val Glu Glu Val Leu Lys Thr Val Ala AsnAsn Arg Asp 435 440 445 aag ggg cgg gcc aac cac agt gcc ttc ctc ttt ggcttt ggg gat ggg 1392 Lys Gly Arg Ala Asn His Ser Ala Phe Leu Phe Gly PheGly Asp Gly 450 455 460 ggt ggt ggc ccc acc cag acc atg ctg gac cgc ctgaag cgc ctg agc 1440 Gly Gly Gly Pro Thr Gln Thr Met Leu Asp Arg Leu LysArg Leu Ser 465 470 475 480 aat acg gat ggg ctg ccc agg gtg cag cta tcttct cca aga cag ctc 1488 Asn Thr Asp Gly Leu Pro Arg Val Gln Leu Ser SerPro Arg Gln Leu 485 490 495 ttc tca gca ctg gag agt gac tca gag cag ctgtgc acg tgg gtt ggg 1536 Phe Ser Ala Leu Glu Ser Asp Ser Glu Gln Leu CysThr Trp Val Gly 500 505 510 gag ctc ttc ttg gag ctg cac aat ggc aca tacacc acc cat gcc cag 1584 Glu Leu Phe Leu Glu Leu His Asn Gly Thr Tyr ThrThr His Ala Gln 515 520 525 atc aag aag ggg aac cgg gaa tgt gag cgg atcctg cac gac gtg gag 1632 Ile Lys Lys Gly Asn Arg Glu Cys Glu Arg Ile LeuHis Asp Val Glu 530 535 540 ctg ctc agt agc ctg gcc ctg gcc cgc agt gcccag ttc cta tac cca 1680 Leu Leu Ser Ser Leu Ala Leu Ala Arg Ser Ala GlnPhe Leu Tyr Pro 545 550 555 560 gca gcc cag ctg cag cac ctc tgg agg ctcctt ctt ctg aac cag ttc 1728 Ala Ala Gln Leu Gln His Leu Trp Arg Leu LeuLeu Leu Asn Gln Phe 565 570 575 cat gat gtg gtg act gga agc tgc atc cagatg gtg gca gag gaa gcc 1776 His Asp Val Val Thr Gly Ser Cys Ile Gln MetVal Ala Glu Glu Ala 580 585 590 atg tgc cat tat gaa gac atc cgt tcc catggc aat aca ctg ctc agc 1824 Met Cys His Tyr Glu Asp Ile Arg Ser His GlyAsn Thr Leu Leu Ser 595 600 605 gct gca gcc gca gcc ctg tgt gct ggg gagcca ggt cct gag ggc ctc 1872 Ala Ala Ala Ala Ala Leu Cys Ala Gly Glu ProGly Pro Glu Gly Leu 610 615 620 ctc atc gtc aac aca ctg ccc tgg aag cggatc gaa gtg atg gcc ctg 1920 Leu Ile Val Asn Thr Leu Pro Trp Lys Arg IleGlu Val Met Ala Leu 625 630 635 640 ccc aaa ccg ggc ggg gcc cac agc ctagcc ctg gtg aca gtg ccc agc 1968 Pro Lys Pro Gly Gly Ala His Ser Leu AlaLeu Val Thr Val Pro Ser 645 650 655 atg ggc tat gct cct gtt cct ccc cccacc tca ctg cag ccc ctg ctg 2016 Met Gly Tyr Ala Pro Val Pro Pro Pro ThrSer Leu Gln Pro Leu Leu 660 665 670 ccc cag cag cct gtg ttc gta gtg caagag act gat ggc tcc gtg act 2064 Pro Gln Gln Pro Val Phe Val Val Gln GluThr Asp Gly Ser Val Thr 675 680 685 ctg gac aat ggc atc atc cga gtg aagctg gac cca act ggt cgc ctg 2112 Leu Asp Asn Gly Ile Ile Arg Val Lys LeuAsp Pro Thr Gly Arg Leu 690 695 700 acg tcc ttg gtc ctg gtg gcc tct ggcagg gag gcc att gct gag ggc 2160 Thr Ser Leu Val Leu Val Ala Ser Gly ArgGlu Ala Ile Ala Glu Gly 705 710 715 720 gcc gtg ggg aac cag ttt gtg ctattt gat gat gtc ccc ttg tac tgg 2208 Ala Val Gly Asn Gln Phe Val Leu PheAsp Asp Val Pro Leu Tyr Trp 725 730 735 gat gca tgg gac gtc atg gac taccac ctg gag aca cgg aag cct gtg 2256 Asp Ala Trp Asp Val Met Asp Tyr HisLeu Glu Thr Arg Lys Pro Val 740 745 750 ctg ggc cag gca ggg acc ctg gcagtg ggc acc gag ggc ggc ctg cgg 2304 Leu Gly Gln Ala Gly Thr Leu Ala ValGly Thr Glu Gly Gly Leu Arg 755 760 765 ggc agc gcc tgg ttc ttg cta cagatc agc ccc aac agt cgg ctt agc 2352 Gly Ser Ala Trp Phe Leu Leu Gln IleSer Pro Asn Ser Arg Leu Ser 770 775 780 cag gag gtt gtg ctg gac gtt ggctgc ccc tat gtc cgc ttc cac acc 2400 Gln Glu Val Val Leu Asp Val Gly CysPro Tyr Val Arg Phe His Thr 785 790 795 800 gag gta cac tgg cat gag gcccac aag ttc ctg aag gtg gag ttc cct 2448 Glu Val His Trp His Glu Ala HisLys Phe Leu Lys Val Glu Phe Pro 805 810 815 gct cgc gtg cgg agt tcc caggcc acc tat gag atc cag ttt ggg cac 2496 Ala Arg Val Arg Ser Ser Gln AlaThr Tyr Glu Ile Gln Phe Gly His 820 825 830 ctg cag cga cct acc cac tacaat acc tct tgg gac tgg gct cga ttt 2544 Leu Gln Arg Pro Thr His Tyr AsnThr Ser Trp Asp Trp Ala Arg Phe 835 840 845 gag gtg tgg gcc cat cgc tggatg gat ctg tca gaa cac ggc ttt ggg 2592 Glu Val Trp Ala His Arg Trp MetAsp Leu Ser Glu His Gly Phe Gly 850 855 860 ctg gcc ctg ctc aac gac tgcaag tat ggc gcg tca gtg cga ggc agc 2640 Leu Ala Leu Leu Asn Asp Cys LysTyr Gly Ala Ser Val Arg Gly Ser 865 870 875 880 atc ctc agc ctc tcg ctcttg cgg gcg cct aaa gcc ccg gac gct act 2688 Ile Leu Ser Leu Ser Leu LeuArg Ala Pro Lys Ala Pro Asp Ala Thr 885 890 895 gct gac acg ggg cgc cacgag ttc acc tat gca ctg atg ccg cac aag 2736 Ala Asp Thr Gly Arg His GluPhe Thr Tyr Ala Leu Met Pro His Lys 900 905 910 ggc tct ttc cag gat gctggc gtt atc caa gct gcc tac agc cta aac 2784 Gly Ser Phe Gln Asp Ala GlyVal Ile Gln Ala Ala Tyr Ser Leu Asn 915 920 925 ttc ccc ctg ttg gct ctgcca gcc ccc agc cca gcg ccc gcc acc tcc 2832 Phe Pro Leu Leu Ala Leu ProAla Pro Ser Pro Ala Pro Ala Thr Ser 930 935 940 tgg agt gcg ttt tcc gtgtct tca ccc gcg gtc gta ttg gag acc gtc 2880 Trp Ser Ala Phe Ser Val SerSer Pro Ala Val Val Leu Glu Thr Val 945 950 955 960 aag cag gcg gag agcagc ccc cag cgc cgc tcg ctg gtc ctg agg ctg 2928 Lys Gln Ala Glu Ser SerPro Gln Arg Arg Ser Leu Val Leu Arg Leu 965 970 975 tat gag gcc cac ggcagc cac gtg gac tgc tgg ctg cac ttg tcg ctg 2976 Tyr Glu Ala His Gly SerHis Val Asp Cys Trp Leu His Leu Ser Leu 980 985 990 ccg gtt cag gag gccatc ctc tgc gat ctc ttg gag cga cca gac cct 3024 Pro Val Gln Glu Ala IleLeu Cys Asp Leu Leu Glu Arg Pro Asp Pro 995 1000 1005 gct ggc cac ttgact tcg gga caa ccg cct gaa gct cac ctt ttc tcc 3072 Ala Gly His Leu ThrSer Gly Gln Pro Pro Glu Ala His Leu Phe Ser 1010 1015 1020 ctt cca agtgct gtc cct gtt gct cgt gct tca gcc tcc gcc aca ctg 3120 Leu Pro Ser AlaVal Pro Val Ala Arg Ala Ser Ala Ser Ala Thr Leu 1025 1030 1035 1040 agtccc tgg ggc tgg ggt ttt gtt tgt aga agg ctc tgg gga ctc cta 3168 Ser ProTrp Gly Trp Gly Phe Val Cys Arg Arg Leu Trp Gly Leu Leu 1045 1050 1055att tct gct tcc cca gcc taa 3189 Ile Ser Ala Ser Pro Ala 1060 60 39 DNAArabidopsis thaliana 60 ggcgcgccct cactctcttc cacttcggcg taccaggac 39 6145 DNA Arabidopsis thaliana 61 ccttaattaa tcacttgtga ggtcgcagttcaagcttata agctc 45 62 42 DNA Caenorhabditis elegans 62 gggcgcgccgcgctcaccaa acgacaagca aatgatttac gg 42 63 44 DNA Caenorhabditis elegans63 gggcgcgccg ctcatattca tcaagtaaag caacatatca agcc 44 64 45 DNACaenorhabditis elegans 64 cttaattaat taaaatgata caagaatact ggaaatatcgtttgg 45 65 36 DNA Ciona intestinalis 65 ggcgcgccac ccttcaagacaaacttagtc tggtgg 36 66 38 DNA Ciona intestinalis 66 ggcgcgccctaccacttata atgcccaagc aatttgcg 38 67 43 DNA Ciona intestinalis 67ccttaattaa ttacgtcagt actattttgt aagcttgtat ctc 43 68 39 DNA Drosophilamelanogaster 68 ggcgcgccca tgagctggaa aatggtttgc aggagcacg 39 69 33 DNADrosophila melanogaster 69 ggcgcgcccg cgacgatcca ataagacctc cac 33 70 38DNA Drosophila melanogaster 70 ggcgcgccga cgtgcccaat gtggatgtac agatgctg38 71 42 DNA Drosophila melanogaster 71 ccttaattaa tcagcttgag tgactgctcacataagcggc gg 42 72 38 DNA Homo sapiens 72 ggcgcgccat agaccatttggagcgtttgc tagctgag 38 73 42 DNA Homo sapiens 73 ggcgcgccgc ttcacaaagtggaagtcaca attcagatgt gc 42 74 44 DNA Homo sapiens 74 cccttaattaatcacctcaa ctggattcgg aatgtgctga tttc 44 75 38 DNA Mus musculus 75ggcgcgccga ccatttggag cgtttgctcg ctgagaac 38 76 32 DNA Mus musculus 76ggcgcgccct gcaggctgac cccagagact gt 32 77 50 DNA Mus musculus 77cccttaatta atcaggtcca acgcaagcgg atacggaacg tgctgatctc 50 78 39 DNARattus norvegicus 78 ggcgcgccgg tgggaacttc cccaggagcc aaatttctg 39 79 41DNA Rattus norvegicus 79 ggcgcgccgc ggagggccca ccagccctgc tgccctacca c41 80 38 DNA Rattus norvegicus 80 ccttaattaa ctaacccaag cgcaggcggaaggtgctg 38 81 36 DNA Homo sapiens 81 ggcgcgccca acacgatccc acccgacaccagaatg 36 82 37 DNA Homo sapiens 82 ggcgcgccgt gctggagctg acagccaacgcagaggg 37 83 39 DNA Homo sapiens 83 ggcgcgccgg tcagaagcca gagctgcagatgctcactg 39 84 41 DNA Homo sapiens 84 ccttaattaa ctaacccaag cggaggcgaaaggtagcaat c 41 85 44 DNA Unknown Organism Description of UnknownOrganism SfMannIII d36 AscI 85 ggcgcgccca gaactataac aaaccaagaatcagttaccc agcc 44 86 47 DNA Unknown Organism Description of UnknownOrganism SfMannIII PacI 86 ccttaattaa ttaaaacctg atcttgtaag tttttacctccatagcg 47 87 39 DNA Homo sapiens 87 ggcgcgccat gggctacgcg cgggcttcgggggtctgcg 39 88 38 DNA Homo sapiens 88 ggcgcgcccc gcctctctgc tttttccttttgttgctg 38 89 41 DNA Homo sapiens 89 ccttaattaa ctaaccatcc acctccttccattgaactga g 41 90 41 DNA Homo sapiens 90 ggcgcgccat ggcggcagcgccgttcttga agcactggcg c 41 91 40 DNA Homo sapiens 91 ccttaattaattaggctggg gaagcagaaa ttaggagtcc 40 92 1143 PRT Caenorhabditis elegans92 Met Gly Lys Arg Asn Phe Tyr Ile Ile Leu Cys Leu Gly Val Phe Leu 1 510 15 Thr Val Ser Leu Tyr Leu Tyr Asn Gly Ile Glu Thr Gly Ala Glu Ala 2025 30 Leu Thr Lys Arg Gln Ala Asn Asp Leu Arg Arg Lys Ile Gly Asn Leu 3540 45 Glu His Val Ala Glu Glu Asn Gly Arg Thr Ile Asp Arg Leu Glu Gln 5055 60 Glu Val Gln Arg Ala Lys Ala Glu Lys Ser Val Asp Phe Asp Glu Glu 6570 75 80 Lys Glu Lys Thr Glu Glu Lys Glu Val Glu Lys Glu Glu Lys Glu Val85 90 95 Ala Pro Val Pro Val Arg Gly Asn Arg Gly Glu Met Ala His Ile His100 105 110 Gln Val Lys Gln His Ile Lys Pro Thr Pro Ser Met Lys Asp ValCys 115 120 125 Gly Ile Arg Glu Asn Val Ser Ile Ala His Ser Asp Leu GlnMet Leu 130 135 140 Asp Leu Tyr Asp Thr Trp Lys Phe Glu Asn Pro Asp GlyGly Val Trp 145 150 155 160 Lys Gln Gly Trp Lys Ile Glu Tyr Asp Ala GluLys Val Lys Ser Leu 165 170 175 Pro Arg Leu Glu Val Ile Val Ile Pro HisSer His Cys Asp Pro Gly 180 185 190 Trp Ile Met Thr Phe Glu Glu Tyr TyrAsn Arg Gln Thr Arg Asn Ile 195 200 205 Leu Asp Gly Met Ala Lys His LeuAla Glu Lys Asp Glu Met Arg Phe 210 215 220 Ile Tyr Ala Glu Ile Ser PhePhe Glu Thr Trp Trp Arg Asp Gln Ala 225 230 235 240 Asp Glu Ile Lys LysLys Val Lys Gly Tyr Leu Glu Ala Gly Lys Phe 245 250 255 Glu Ile Val ThrGly Gly Trp Val Met Thr Asp Glu Ala Asn Ala His 260 265 270 Tyr His SerMet Ile Thr Glu Leu Phe Glu Gly His Glu Trp Ile Gln 275 280 285 Asn HisLeu Gly Lys Ser Ala Ile Pro Gln Ser His Trp Ser Ile Asp 290 295 300 ProPhe Gly Leu Ser Pro Ser Met Pro His Leu Leu Thr Ser Ala Asn 305 310 315320 Ile Thr Asn Ala Val Ile Gln Arg Val His Tyr Ser Val Lys Arg Glu 325330 335 Leu Ala Leu Lys Lys Asn Leu Glu Phe Tyr Trp Arg Gln Leu Phe Gly340 345 350 Ser Thr Gly His Pro Asp Leu Arg Ser His Ile Met Pro Phe TyrSer 355 360 365 Tyr Asp Ile Pro His Thr Cys Gly Pro Glu Pro Ser Val CysCys Gln 370 375 380 Phe Asp Phe Arg Arg Met Pro Glu Gly Gly Lys Ser CysAsp Trp Gly 385 390 395 400 Ile Pro Pro Gln Lys Ile Asn Asp Asp Asn ValAla His Arg Ala Glu 405 410 415 Met Ile Tyr Asp Gln Tyr Arg Lys Lys SerGln Leu Phe Lys Asn Asn 420 425 430 Val Ile Phe Gln Pro Leu Gly Asp AspPhe Arg Tyr Asp Ile Asp Phe 435 440 445 Glu Trp Asn Ser Gln Tyr Glu AsnTyr Lys Lys Leu Phe Glu Tyr Met 450 455 460 Asn Ser Lys Ser Glu Trp AsnVal His Ala Gln Phe Gly Thr Leu Ser 465 470 475 480 Asp Tyr Phe Lys LysLeu Asp Thr Ala Ile Ser Ala Ser Gly Glu Gln 485 490 495 Leu Pro Thr PheSer Gly Asp Phe Phe Thr Tyr Ala Asp Arg Asp Asp 500 505 510 His Tyr TrpSer Gly Tyr Phe Thr Ser Arg Pro Phe Tyr Lys Gln Leu 515 520 525 Asp ArgVal Leu Gln His Tyr Leu Arg Ser Ala Glu Ile Ala Phe Thr 530 535 540 LeuAla Asn Ile Glu Glu Glu Gly Met Val Glu Ala Lys Ile Phe Glu 545 550 555560 Lys Leu Val Thr Ala Arg Arg Ala Leu Ser Leu Phe Gln His His Asp 565570 575 Gly Val Thr Gly Thr Ala Lys Asp His Val Val Leu Asp Tyr Gly Gln580 585 590 Lys Met Ile Asp Ala Leu Asn Ala Cys Glu Asp Ile Leu Ser GluAla 595 600 605 Leu Val Val Leu Leu Gly Ile Asp Ser Thr Asn Lys Met GlnMet Asp 610 615 620 Glu His Arg Val Asn Glu Asn Leu Leu Pro Glu Lys ArgVal Tyr Lys 625 630 635 640 Ile Gly Gln Asn Val Val Leu Phe Asn Thr LeuSer Arg Asn Arg Asn 645 650 655 Glu Pro Ile Cys Ile Gln Val Asp Ser LeuAsp Ala Gly Val Glu Ala 660 665 670 Asp Pro Pro Ile Lys Lys Gln Gln ValSer Pro Val Ile Ala Tyr Asp 675 680 685 Glu Glu Lys Lys Thr Leu Val ValLys Asn Gly Ile Phe Glu Leu Cys 690 695 700 Phe Met Leu Ser Leu Gly ProMet Glu Ser Val Ser Phe Arg Leu Val 705 710 715 720 Lys Asn Thr Thr ThrSer Lys Val Glu Ile Ile Thr Asn Asn Ala Ala 725 730 735 Glu Phe Lys GluThr Ser Phe Lys Ser Ser Ser Thr Ser Gly Asp Phe 740 745 750 Thr Val LysAsn Asp Lys Val Glu Ala Glu Phe Asp Gly Glu Asn Gly 755 760 765 Met IleLys Arg Ala Thr Ser Leu Val Asp Asp Lys Pro Ile Asp Leu 770 775 780 AsnSer His Phe Ile His Tyr Gly Ala Arg Lys Ser Lys Arg Lys Phe 785 790 795800 Ala Asn Gly Asn Glu Asp Asn Pro Ala Gly Ala Tyr Leu Phe Leu Pro 805810 815 Asp Gly Glu Ala Arg Glu Leu Lys Lys Gln Ser Ser Asp Trp Ile Leu820 825 830 Val Lys Gly Glu Val Val Gln Lys Val Phe Ala Thr Pro Asn AsnAsp 835 840 845 Leu Lys Ile Leu Gln Thr Tyr Thr Leu Tyr Gln Gly Leu ProTrp Ile 850 855 860 Asp Leu Asp Asn Glu Val Asp Val Arg Ser Lys Glu AsnPhe Glu Leu 865 870 875 880 Ala Leu Arg Phe Ser Ser Ser Val Asn Ser GlyAsp Glu Phe Phe Thr 885 890 895 Asp Leu Asn Gly Met Gln Met Ile Lys ArgArg Arg Gln Thr Lys Leu 900 905 910 Pro Thr Gln Ala Asn Phe Tyr Pro MetSer Ala Gly Val Tyr Ile Glu 915 920 925 Asp Asp Thr Thr Arg Met Ser IleHis Ser Ala Gln Ala Leu Gly Val 930 935 940 Ser Ser Leu Ser Ser Gly GlnIle Glu Ile Met Leu Asp Arg Arg Leu 945 950 955 960 Ser Ser Asp Asp AsnArg Gly Leu Gln Gln Gly Val Arg Asp Asn Lys 965 970 975 Arg Thr Val AlaHis Phe Arg Ile Val Ile Glu Pro Met Ser Ser Ser 980 985 990 Ser Gly AsnLys Lys Glu Glu Arg Val Gly Phe His Ser His Val Gly 995 1000 1005 HisLeu Ala Thr Trp Ser Leu His Tyr Pro Leu Val Lys Met Ile Gly 1010 10151020 Asp Ala Thr Pro Lys Ser Ile Ser Ser Lys Asn Val Glu Gln Glu Leu1025 1030 1035 1040 Asn Cys Asp Leu His Leu Val Thr Phe Arg Thr Leu AlaSer Pro Thr 1045 1050 1055 Thr Tyr Glu Ala Asn Glu Arg Ser Thr Ala AlaGlu Lys Lys Ala Ala 1060 1065 1070 Met Val Met His Arg Val Val Pro AspCys Arg Ser Arg Leu Thr Leu 1075 1080 1085 Pro Asp Thr Ser Cys Leu AlaThr Gly Leu Glu Ile Glu Pro Leu Lys 1090 1095 1100 Leu Ile Ser Thr LeuLys Ser Ala Lys Lys Thr Ser Leu Thr Asn Leu 1105 1110 1115 1120 Tyr GluGly Asn Lys Ala Glu Gln Phe Arg Leu Gln Pro Asn Asp Ile 1125 1130 1135Ser Ser Ile Leu Val Ser Phe 1140 93 1279 PRT Rattus norvegicus 93 MetAla Cys Ile Gly Gly Ala Gln Gly Gln Arg Gln Ala Val Glu Lys 1 5 10 15Glu Pro Ser His Gln Gly Tyr Pro Trp Lys Pro Met Thr Asn Gly Ser 20 25 30Cys Ser Glu Leu Ala Leu Leu Ser Lys Thr Arg Met Tyr Cys His Gln 35 40 45Gly Cys Val Arg Pro Pro Arg Thr Asp Val Lys Asn Phe Lys Thr Thr 50 55 60Thr Asp Thr Gln Ser Val Pro Gly Val Ser Met Lys Leu Lys Lys Gln 65 70 7580 Val Thr Val Cys Gly Ala Ala Ile Phe Cys Val Ala Val Phe Ser Leu 85 9095 Tyr Leu Met Leu Asp Arg Val Gln His Asp Pro Ala Arg His Gln Asn 100105 110 Gly Gly Asn Phe Pro Arg Ser Gln Ile Ser Val Leu Gln Asn Arg Ile115 120 125 Glu Gln Leu Glu Gln Leu Leu Glu Glu Asn His Glu Ile Ile SerHis 130 135 140 Ile Lys Asp Ser Val Leu Glu Leu Thr Ala Asn Ala Glu GlyPro Pro 145 150 155 160 Ala Leu Leu Pro Tyr His Thr Ala Asn Gly Ser TrpAla Val Leu Pro 165 170 175 Glu Pro Arg Pro Ser Phe Phe Ser Val Ser ProGlu Asp Cys Gln Phe 180 185 190 Ala Leu Gly Gly Arg Gly Gln Lys Pro GluLeu Gln Met Leu Thr Val 195 200 205 Ser Glu Asp Leu Pro Phe Asp Asn ValGlu Gly Gly Val Trp Arg Gln 210 215 220 Gly Phe Asp Ile Ser Tyr Ser ProAsn Asp Trp Asp Ala Glu Asp Leu 225 230 235 240 Gln Val Phe Val Val ProHis Ser His Asn Asp Pro Gly Glu Glu Pro 245 250 255 Ala Gly Pro Ser ArgSer Val Gln Gly Gly Leu Ser Gly Asp Arg Arg 260 265 270 Trp Ile Lys ThrPhe Asp Lys Tyr Tyr Thr Glu Gln Thr Gln His Ile 275 280 285 Leu Asn SerMet Val Ser Lys Leu Gln Glu Asp Pro Arg Arg Arg Phe 290 295 300 Leu TrpAla Glu Val Ser Phe Phe Ala Lys Trp Trp Asp Asn Ile Ser 305 310 315 320Ala Gln Lys Arg Ala Ala Val Arg Arg Leu Val Gly Asn Gly Gln Leu 325 330335 Glu Ile Ala Thr Gly Gly Trp Val Met Pro Asp Glu Ala Asn Ser His 340345 350 Tyr Phe Ala Leu Val Gly Gln Leu Ile Glu Gly Pro Pro Pro Val Arg355 360 365 Arg Ala Val Asp Pro Phe Gly His Ser Ser Thr Met Pro Tyr LeuLeu 370 375 380 Arg Arg Ala Asn Leu Thr Ser Met Leu Ile Gln Arg Val HisTyr Ala 385 390 395 400 Ile Lys Lys His Phe Ala Ala Thr His Ser Leu GluPhe Met Trp Arg 405 410 415 Gln Thr Trp Asp Ser Asp Ser Ser Thr Asp IlePhe Cys His Met Met 420 425 430 Pro Phe Tyr Ser Tyr Asp Val Pro His ThrCys Gly Pro Asp Pro Lys 435 440 445 Ile Cys Cys Gln Phe Asp Phe Lys ArgLeu Pro Gly Gly Arg Ile Asn 450 455 460 Cys Pro Trp Lys Val Pro Pro ArgAla Ile Thr Glu Ala Asn Val Ala 465 470 475 480 Asp Arg Ala Ala Leu LeuLeu Asp Gln Tyr Arg Lys Lys Ser Arg Leu 485 490 495 Phe Arg Ser Ser ValLeu Leu Val Pro Leu Gly Asp Asp Phe Arg Tyr 500 505 510 Asp Lys Pro GlnGlu Trp Asp Ala Gln Phe Phe Asn Tyr Gln Arg Leu 515 520 525 Phe Asp PheLeu Asn Ser Lys Pro Glu Phe His Val Gln Ala Gln Phe 530 535 540 Gly ThrLeu Ser Glu Tyr Phe Asp Ala Leu Tyr Lys Arg Thr Gly Val 545 550 555 560Glu Pro Gly Ala Arg Pro Pro Gly Phe Pro Val Leu Ser Gly Asp Phe 565 570575 Phe Ser Tyr Ala Asp Arg Glu Asp His Tyr Trp Thr Gly Tyr Tyr Thr 580585 590 Ser Arg Pro Phe Tyr Lys Ser Leu Asp Arg Val Leu Glu Thr His Leu595 600 605 Arg Gly Ala Glu Val Leu Tyr Ser Leu Ala Leu Ala His Ala ArgArg 610 615 620 Ser Gly Leu Thr Gly Gln Tyr Pro Leu Ser Asp Tyr Ala ValLeu Thr 625 630 635 640 Glu Ala Arg Arg Thr Leu Gly Leu Phe Gln His HisAsp Ala Ile Thr 645 650 655 Gly Thr Ala Lys Glu Ala Val Val Val Asp TyrGly Val Arg Leu Leu 660 665 670 Arg Ser Leu Val Ser Leu Lys Gln Val IleIle Asn Ala Ala His Tyr 675 680 685 Leu Val Leu Gly Asp Lys Glu Thr TyrSer Phe Asp Pro Arg Ala Pro 690 695 700 Phe Leu Gln Met Val Ser Gln AlaTrp Arg Gly Ser Gln Ser Thr Leu 705 710 715 720 His Pro Ser Ala Ala LeuVal Pro Ala Ala Ala Ala Ser Ala Leu Leu 725 730 735 Pro Gln Arg Ala ProArg Phe Val Val Val Phe Asn Pro Leu Glu Gln 740 745 750 Glu Arg Leu SerVal Val Ser Leu Leu Val Asn Ser Pro Arg Val Arg 755 760 765 Val Leu SerGlu Glu Gly Gln Pro Leu Ser Val Gln Ile Ser Val Gln 770 775 780 Trp SerSer Ala Thr Asn Met Val Pro Asp Val Tyr Gln Val Ser Val 785 790 795 800Pro Val Arg Leu Pro Ala Leu Gly Leu Gly Val Leu Gln Leu Gln Pro 805 810815 Asp Leu Asp Gly Pro Tyr Thr Leu Gln Ser Ser Val His Val Tyr Leu 820825 830 Asn Gly Val Lys Leu Ser Val Ser Arg Gln Thr Thr Phe Pro Leu Arg835 840 845 Val Val Asp Ser Gly Thr Ser Asp Phe Ala Ile Ser Asn Arg TyrMet 850 855 860 Gln Val Trp Phe Ser Gly Leu Thr Gly Leu Leu Lys Ser ValArg Arg 865 870 875 880 Val Asp Glu Glu Gln Glu Gln Gln Val Asp Met LysLeu Phe Val Tyr 885 890 895 Gly Thr Arg Thr Ser Lys Asp Lys Ser Gly AlaTyr Leu Phe Leu Pro 900 905 910 Asp Asn Glu Ala Lys Pro Tyr Val Pro LysLys Pro Pro Val Leu Arg 915 920 925 Val Thr Glu Gly Pro Phe Phe Ser GluVal Ala Ala Tyr Tyr Glu His 930 935 940 Phe His Gln Val Ile Arg Leu TyrAsn Leu Pro Gly Val Glu Gly Leu 945 950 955 960 Ser Leu Asp Val Ser PheGln Val Asp Ile Arg Asp Tyr Val Asn Lys 965 970 975 Glu Leu Ala Leu ArgIle His Thr Asp Ile Asp Ser Gln Gly Thr Phe 980 985 990 Phe Thr Asp LeuAsn Gly Phe Gln Val Gln Pro Arg Lys Tyr Leu Lys 995 1000 1005 Lys LeuPro Leu Gln Ala Asn Phe Tyr Pro Met Pro Val Met Ala Tyr 1010 1015 1020Ile Gln Asp Ser Gln Arg Arg Leu Thr Leu His Thr Ala Gln Ala Leu 10251030 1035 1040 Gly Val Ser Ser Leu Gly Asn Gly Gln Leu Glu Val Ile LeuAsp Arg 1045 1050 1055 Arg Leu Met Gln Asp Asp Asn Arg Gly Leu Gly GlnGly Leu Lys Asp 1060 1065 1070 Asn Lys Ile Thr Cys Asn His Phe Arg LeuLeu Leu Glu Arg Arg Thr 1075 1080 1085 Leu Met Ser Pro Glu Val Gln GlnGlu Arg Ser Thr Ser Tyr Pro Ser 1090 1095 1100 Leu Leu Ser His Met ThrSer Met Tyr Leu Asn Thr Pro Pro Leu Val 1105 1110 1115 1120 Leu Pro ValAla Lys Arg Glu Ser Thr Ser Pro Thr Leu His Ser Phe 1125 1130 1135 HisPro Leu Ala Ser Pro Leu Pro Cys Asp Phe His Leu Leu Asn Leu 1140 11451150 Arg Met Leu Pro Ala Glu Val Ser Val Pro Val Arg Ala Asn Pro His1155 1160 1165 His Gln Ala Glu Pro Cys Leu Leu Gly Arg His Ala Ala AspPro Pro 1170 1175 1180 Pro Leu Leu Ser Leu Thr Val Phe Gln Asp Thr LeuPro Ala Ala Asp 1185 1190 1195 1200 Ala Ala Leu Ile Leu His Arg Lys GlyPhe Asp Cys Gly Leu Glu Ala 1205 1210 1215 Lys Asn Leu Gly Phe Asn CysThr Thr Ser Gln Gly Lys Leu Ala Leu 1220 1225 1230 Gly Ser Leu Phe HisGly Leu Asp Val Leu Phe Leu Gln Pro Thr Ser 1235 1240 1245 Leu Thr LeuLeu Tyr Pro Leu Ala Ser Pro Ser Asn Ser Thr Asp Ile 1250 1255 1260 SerLeu Glu Pro Met Glu Ile Ser Thr Phe Arg Leu Arg Leu Gly 1265 1270 127594 1149 PRT Ciona intestinalis 94 Met Lys Leu Lys Arg Gln Phe Leu PhePhe Gly Gly Ile Leu Phe Phe 1 5 10 15 Gly Ser Ile Trp Phe Met Ile GlyGln Leu Asp Thr Pro Asn Ser Pro 20 25 30 Gln Lys Val Lys Phe Ser Glu GlySer Glu Asn Asp Gln Val Arg Thr 35 40 45 Leu Gln Asp Lys Leu Ser Leu ValGlu Lys Glu Leu Leu Glu Asn Arg 50 55 60 Lys Ile Met His Lys Val Lys AspSer Leu Gln Asp Met Thr Pro Met 65 70 75 80 Lys Asn Val His Val Pro MetGln Arg Gly Glu Ile Arg Asn Asn Val 85 90 95 Asn Lys Pro Val Leu Pro LeuIle Met Pro Lys Gln Phe Ala Asn Asp 100 105 110 Ser Arg Met Ser Asp ThrCys Pro Val Leu Ser Tyr Ser Gly Gly Lys 115 120 125 Ser Asp Val Asn MetIle Asn Val Tyr Asp His Leu Pro Phe Asp Asp 130 135 140 Pro Asp Gly GlyVal Trp Lys Gln Gly Trp Asp Ile Gln Thr Ser Asp 145 150 155 160 Gln GluTrp Ala Gly Arg Lys Leu Lys Val Phe Ile Val Pro His Ser 165 170 175 HisAsn Asp Pro Gly Trp Leu Lys Thr Val Glu Arg Tyr Phe Ser Asp 180 185 190Gln Thr Gln His Ile Leu Asn Asn Ile Val Asp Ala Leu Ser Gln Asp 195 200205 Pro Ala Arg Lys Phe Ile Trp Ala Glu Met Ser Tyr Leu Ser Met Trp 210215 220 Trp Asp Ile Ala Thr Pro Asp Arg Lys Gln Lys Met Gln Thr Leu Val225 230 235 240 Lys Asn Gly Gln Leu Glu Ile Val Thr Gly Gly Trp Val MetAsn Asp 245 250 255 Glu Ala Asn Thr His Tyr Phe Ala Met Ile Asp Gln LeuIle Glu Gly 260 265 270 Met Glu Trp Leu Arg Arg Thr Leu Asn Val Val ProLys Ser Gly Trp 275 280 285 Ala Ile Asp Pro Phe Gly His Thr Pro Thr MetAla Tyr Ile Leu Lys 290 295 300 Gln Met Lys Phe Lys Asn Met Leu Ile GlnArg Val His Tyr Ala Val 305 310 315 320 Lys Lys Tyr Leu Ala Gln Glu LysSer Leu Glu Phe Arg Trp Arg Gln 325 330 335 Met Trp Asp Ser Ala Ser SerThr Asp Met Met Cys His Leu Met Pro 340 345 350 Phe Tyr Ser Tyr Asp ValPro His Thr Cys Gly Pro Asp Pro Lys Ile 355 360 365 Cys Cys Gln Phe AspPhe Ala Arg Leu Pro Gly Gly Lys Ile Thr Cys 370 375 380 Pro Trp Lys ValPro Pro Val Ala Ile Thr Asp Ser Asn Val Glu Thr 385 390 395 400 Arg AlaGly Ile Leu Leu Asp Gln Tyr Arg Lys Lys Ser Lys Leu Phe 405 410 415 LysSer Asp Thr Leu Leu Ile Ile Leu Gly Asp Asp Phe Arg Tyr Ser 420 425 430Leu Ser Lys Glu Thr Asn Asp Gln Phe Asp Asn Tyr Ala Arg Ile Ile 435 440445 Ser Tyr Val Asn Ser His Pro Glu Leu Asn Ala Lys Leu Gln Phe Gly 450455 460 Thr Leu Ser Glu Tyr Phe Asp Ala Met Lys Ser Glu Val Gly Gly Glu465 470 475 480 Glu Lys Leu Pro Ala Leu Ser Gly Asp Phe Phe Thr Tyr AlaAsp Arg 485 490 495 Glu Asp His Tyr Trp Ser Gly Tyr Tyr Thr Ser Arg ProTyr His Lys 500 505 510 Met Gln Glu Arg Val Leu Glu Ser His Leu Arg GlyAla Glu Met Leu 515 520 525 Phe Ala Leu Ser Trp Pro Lys Ile Gln Trp ThrGly Leu Gly Glu Thr 530 535 540 Phe Ser His Glu Leu Tyr Pro Leu Leu ValGln Ala Arg Gln Asn Leu 545 550 555 560 Gly Leu Phe Gln His His Asp GlyIle Thr Gly Thr Ala Lys Asp His 565 570 575 Val Val Val Asp Tyr Gly AsnLys Leu Met Lys Ser Val Met Asp Ala 580 585 590 Lys Lys Val Ile Ser TyrSer Ala Gln Val Leu Leu Gln Glu Met Ile 595 600 605 Thr Phe Asp Pro AsnThr Met Val Leu Asn Tyr Asp Glu Val Tyr Gln 610 615 620 Ala Gln Asn GlnGln Pro Ala Pro Val Val Val Lys Leu Pro Thr Lys 625 630 635 640 Asn GluGlu Ala Arg Lys Val Val Leu Tyr Asn Ser Leu Asp Tyr Asp 645 650 655 ArgThr Gly Val Val Arg Leu Ile Val Thr Ser Pro Asp Val Val Val 660 665 670Met Ser Glu Asn Lys Asn Val Val Pro Ser Gln Thr Ser Pro Ile Trp 675 680685 Ser Asp Ser Thr Glu Ile Arg Thr Asp Gln Phe Glu Leu Val Phe Leu 690695 700 Ser Thr Val Pro Ala Ile Gly Leu Ala Val Tyr Lys Ile Trp Glu Asp705 710 715 720 Asn Asp Val Ala Asp Thr Thr His Ser Thr Val Lys Phe IleAsn Pro 725 730 735 Arg Val Gly Phe Ser Lys Arg Thr Arg Ser Lys Phe ValLeu Asp Val 740 745 750 Glu Asp Ser Gly Glu Phe Thr Ile Met Asn Asp GlnLeu Val Ala His 755 760 765 Phe Ser Gly Gln Asn Gly Met Leu Gln Ser ValThr Thr Val Arg Asp 770 775 780 Asn Val Lys Thr Gln Leu Gly Ile Glu PheVal Ala Tyr Thr Ser Arg 785 790 795 800 Asn Lys Lys Asp Lys Ser Gly AlaTyr Leu Phe Leu Pro Ala Gly Pro 805 810 815 Ala Gln Pro His Val Thr GluSer His Arg Pro Leu Val Arg Ile Ile 820 825 830 Arg Gly Pro Val Met SerThr Val His Val Leu Leu Pro Asn Val Leu 835 840 845 His Lys Val Thr LeuTyr Thr Gly Thr Gly Ala Gly Thr Gln Ser Leu 850 855 860 Gly Val His ValSer Asn Asp Val Asp Val Arg Thr Gly Tyr Asp Asn 865 870 875 880 Lys GluLeu Ser Met Arg Leu Asn Ser Glu Val Leu Ser Gly Ser Lys 885 890 895 PhePhe Thr Asp Leu Asn Gly Phe Gln Ile Gln Pro Arg Thr Thr Tyr 900 905 910Ser Lys Leu Pro Leu Gln Ala Asn Phe Tyr Pro Ile Pro Thr Met Ala 915 920925 Phe Ile Gln Asp Glu Lys Ser Arg Leu Thr Leu Met Thr Ala Gln Pro 930935 940 Leu Gly Val Ala Ser Leu Lys Ser Gly Gln Leu Glu Val Val Leu Asp945 950 955 960 Arg Arg Leu Met Gln Asp Asp Asn Arg Gly Val Gly Gln GlyVal Lys 965 970 975 Asp Asn Leu Pro Thr Pro Glu Ser Phe Val Ile Met LeuGlu Arg Trp 980 985 990 Thr Ala Ile Ala Ala Lys Glu Ser Lys Ser Ser AlaLys Leu Ala Tyr 995 1000 1005 Pro Ser Met Ala Val Tyr Gln Ser Ser TrpGlu Leu Leu His Pro Ile 1010 1015 1020 Arg Pro Met Ser Val Asn Gly ProVal His Leu Lys Glu Asp Tyr Arg 1025 1030 1035 1040 Ser Leu Pro Gln ProLeu Pro Cys Asp Val His Val Leu Asn Leu Arg 1045 1050 1055 Ala Ile HisSer Lys Asp Ala Val Ala Pro Thr Asp Gln Ser Ala Leu 1060 1065 1070 LeuLeu His Thr Val Gly Arg Glu Cys Ser Leu Asp Ala Asp Lys Tyr 1075 10801085 Phe His Pro Thr Cys Leu Met His Gly Val Glu Lys Leu Ala Ile Thr1090 1095 1100 Ile Ser Thr Leu Phe Thr Asn Ser Gly Met Arg Lys Thr SerLeu Ser 1105 1110 1115 1120 Leu Gln His Asp Gly Ser Leu Leu Asp Asn GlnGly Gly Ile Thr Val 1125 1130 1135 Ser Pro Met Glu Ile Gln Ala Tyr LysIle Val Leu Thr 1140 1145 95 1173 PRT Arabidopsis thaliana 95 Met ProPhe Ser Ser Tyr Ile Gly Asn Ser Arg Arg Ser Ser Thr Gly 1 5 10 15 GlyGly Thr Gly Gly Trp Gly Gln Ser Leu Leu Pro Thr Ala Leu Ser 20 25 30 LysSer Lys Leu Ala Ile Asn Arg Lys Pro Arg Lys Arg Thr Leu Val 35 40 45 ValAsn Phe Ile Phe Ala Asn Phe Phe Val Ile Ala Leu Thr Val Ser 50 55 60 LeuLeu Phe Phe Leu Leu Thr Leu Phe His Phe Gly Val Pro Gly Pro 65 70 75 80Ile Ser Ser Arg Phe Leu Thr Ser Arg Ser Asn Arg Ile Val Lys Pro 85 90 95Arg Lys Asn Ile Asn Arg Arg Pro Leu Asn Asp Ser Asn Ser Gly Ala 100 105110 Val Val Asp Ile Thr Thr Lys Asp Leu Tyr Asp Arg Ile Glu Phe Leu 115120 125 Asp Thr Asp Gly Gly Pro Trp Lys Gln Gly Trp Arg Val Thr Tyr Lys130 135 140 Asp Asp Glu Trp Glu Lys Glu Lys Leu Lys Ile Phe Val Val ProHis 145 150 155 160 Ser His Asn Asp Pro Gly Trp Lys Leu Thr Val Glu GluTyr Tyr Gln 165 170 175 Arg Gln Ser Arg His Ile Leu Asp Thr Ile Val GluThr Leu Ser Lys 180 185 190 Asp Ser Arg Arg Lys Phe Ile Trp Glu Glu MetSer Tyr Leu Glu Arg 195 200 205 Trp Trp Arg Asp Ala Ser Pro Asn Lys GlnGlu Ala Leu Thr Lys Leu 210 215 220 Val Lys Asp Gly Gln Leu Glu Ile ValGly Gly Gly Trp Val Met Asn 225 230 235 240 Asp Glu Ala Asn Ser His TyrPhe Ala Ile Ile Glu Gln Ile Ala Glu 245 250 255 Gly Asn Met Trp Leu AsnAsp Thr Ile Gly Val Ile Pro Lys Asn Ser 260 265 270 Trp Ala Ile Asp ProPhe Gly Tyr Ser Ser Thr Met Ala Tyr Leu Leu 275 280 285 Arg Arg Met GlyPhe Glu Asn Met Leu Ile Gln Arg Thr His Tyr Glu 290 295 300 Leu Lys LysAsp Leu Ala Gln His Lys Asn Leu Glu Tyr Ile Trp Arg 305 310 315 320 GlnSer Trp Asp Ala Met Glu Thr Thr Asp Ile Phe Val His Met Met 325 330 335Pro Phe Tyr Ser Tyr Asp Ile Pro His Thr Cys Gly Pro Glu Pro Ala 340 345350 Ile Cys Cys Gln Phe Asp Phe Ala Arg Met Arg Gly Phe Lys Tyr Glu 355360 365 Leu Cys Pro Trp Gly Lys His Pro Val Glu Thr Thr Leu Glu Asn Val370 375 380 Gln Glu Arg Ala Leu Lys Leu Leu Asp Gln Tyr Arg Lys Lys SerThr 385 390 395 400 Leu Tyr Arg Thr Asn Thr Leu Leu Ile Pro Leu Gly AspAsp Phe Arg 405 410 415 Tyr Ile Ser Ile Asp Glu Ala Glu Ala Gln Phe ArgAsn Tyr Gln Met 420 425 430 Leu Phe Asp His Ile Asn Ser Asn Pro Ser LeuAsn Ala Glu Ala Lys 435 440 445 Phe Gly Thr Leu Glu Asp Tyr Phe Arg ThrVal Arg Glu Glu Ala Asp 450 455 460 Arg Val Asn Tyr Ser Arg Pro Gly GluVal Gly Ser Gly Gln Val Val 465 470 475 480 Gly Phe Pro Ser Leu Ser GlyAsp Phe Phe Thr Tyr Ala Asp Arg Gln 485 490 495 Gln Asp Tyr Trp Ser GlyTyr Tyr Val Ser Arg Pro Phe Phe Lys Ala 500 505 510 Val Asp Arg Val LeuGlu His Thr Leu Arg Gly Ala Glu Ile Met Met 515 520 525 Ser Phe Leu LeuGly Tyr Cys His Arg Ile Gln Cys Glu Lys Phe Pro 530 535 540 Thr Ser PheThr Tyr Lys Leu Thr Ala Ala Arg Arg Asn Leu Ala Leu 545 550 555 560 PheGln His His Asp Gly Val Thr Gly Thr Ala Lys Asp Tyr Val Val 565 570 575Gln Asp Tyr Gly Thr Arg Met His Thr Ser Leu Gln Asp Leu Gln Ile 580 585590 Phe Met Ser Lys Ala Ile Glu Val Leu Leu Gly Ile Arg His Glu Lys 595600 605 Glu Lys Ser Asp Gln Ser Pro Ser Phe Phe Glu Ala Glu Gln Met Arg610 615 620 Ser Lys Tyr Asp Ala Arg Pro Val His Lys Pro Ile Ala Ala ArgGlu 625 630 635 640 Gly Asn Ser His Thr Val Ile Leu Phe Asn Pro Ser GluGln Thr Arg 645 650 655 Glu Glu Val Val Thr Val Val Val Asn Arg Ala GluIle Ser Val Leu 660 665 670 Asp Ser Asn Trp Thr Cys Val Pro Ser Gln IleSer Pro Glu Val Gln 675 680 685 His Asp Asp Thr Lys Leu Phe Thr Gly ArgHis Arg Leu Tyr Trp Lys 690 695 700 Ala Ser Ile Pro Ala Leu Gly Leu ArgThr Tyr Phe Ile Ala Asn Gly 705 710 715 720 Asn Val Glu Cys Glu Lys AlaThr Pro Ser Lys Leu Lys Tyr Ala Ser 725 730 735 Glu Phe Asp Pro Phe ProCys Pro Pro Pro Tyr Ser Cys Ser Lys Leu 740 745 750 Asp Asn Asp Val ThrGlu Ile Arg Asn Glu His Gln Thr Leu Val Phe 755 760 765 Asp Val Lys AsnGly Ser Leu Arg Lys Ile Val His Arg Asn Gly Ser 770 775 780 Glu Thr ValVal Gly Glu Glu Ile Gly Met Tyr Ser Ser Pro Glu Ser 785 790 795 800 GlyAla Tyr Leu Phe Lys Pro Asp Gly Glu Ala Gln Pro Ile Val Gln 805 810 815Pro Asp Gly His Val Val Thr Ser Glu Gly Leu Leu Val Gln Glu Val 820 825830 Phe Ser Tyr Pro Lys Thr Lys Trp Glu Lys Ser Pro Leu Ser Gln Lys 835840 845 Thr Arg Leu Tyr Thr Gly Gly Asn Thr Leu Gln Asp Gln Val Val Glu850 855 860 Ile Glu Tyr His Val Glu Leu Leu Gly Asn Asp Phe Asp Asp ArgGlu 865 870 875 880 Leu Ile Val Arg Tyr Lys Thr Asp Val Asp Asn Lys LysVal Phe Tyr 885 890 895 Ser Asp Leu Asn Gly Phe Gln Met Ser Arg Arg GluThr Tyr Asp Lys 900 905 910 Ile Pro Leu Gln Gly Asn Tyr Tyr Pro Met ProSer Leu Ala Phe Ile 915 920 925 Gln Gly Ser Asn Gly Gln Arg Phe Ser ValHis Ser Arg Gln Ser Leu 930 935 940 Gly Val Ala Ser Leu Lys Glu Gly TrpLeu Glu Ile Met Leu Asp Arg 945 950 955 960 Arg Leu Val Arg Asp Asp GlyArg Gly Leu Gly Gln Gly Val Met Asp 965 970 975 Asn Arg Ala Met Thr ValVal Phe His Leu Leu Ala Glu Ser Asn Ile 980 985 990 Ser Gln Ala Asp ProAla Ser Asn Thr Asn Pro Arg Asn Pro Ser Leu 995 1000 1005 Leu Ser HisLeu Ile Gly Ala His Leu Asn Tyr Pro Ile Asn Thr Phe 1010 1015 1020 IleAla Lys Lys Pro Gln Asp Ile Ser Val Arg Val Pro Gln Tyr Gly 1025 10301035 1040 Ser Phe Ala Pro Leu Ala Lys Pro Leu Pro Cys Asp Leu His IleVal 1045 1050 1055 Asn Phe Lys Val Pro Arg Pro Ser Lys Tyr Ser Gln GlnLeu Glu Glu 1060 1065 1070 Asp Lys Pro Arg Phe Ala Leu Ile Leu Asn ArgArg Ala Trp Asp Ser 1075 1080 1085 Ala Tyr Cys His Lys Gly Arg Gln ValAsn Cys Thr Ser Met Ala Asn 1090 1095 1100 Glu Pro Val Asn Phe Ser AspMet Phe Lys Asp Leu Ala Ala Ser Lys 1105 1110 1115 1120 Val Lys Pro ThrSer Leu Asn Leu Leu Gln Glu Asp Met Glu Ile Leu 1125 1130 1135 Gly TyrAsp Asp Gln Glu Leu Pro Arg Asp Ser Ser Gln Pro Arg Glu 1140 1145 1150Gly Arg Val Ser Ile Ser Pro Met Glu Ile Arg Ala Tyr Lys Leu Glu 11551160 1165 Leu Arg Pro His Lys 1170 96 1108 PRT Drosophila sp. 96 Met LeuArg Ile Arg Arg Arg Phe Ala Leu Val Ile Cys Ser Gly Cys 1 5 10 15 LeuLeu Val Phe Leu Ser Leu Tyr Ile Ile Leu Asn Phe Ala Ala Pro 20 25 30 AlaAla Thr Gln Ile Lys Pro Asn Tyr Glu Asn Ile Glu Asn Lys Leu 35 40 45 HisGlu Leu Glu Asn Gly Leu Gln Glu His Gly Glu Glu Met Arg Asn 50 55 60 LeuArg Ala Arg Leu Ala Lys Thr Ser Asn Arg Asp Asp Pro Ile Arg 65 70 75 80Pro Pro Leu Lys Val Ala Arg Ser Pro Arg Pro Gly Gln Cys Gln Asp 85 90 95Val Val Gln Asp Val Pro Asn Val Asp Val Gln Met Leu Glu Leu Tyr 100 105110 Asp Arg Met Ser Phe Lys Asp Ile Asp Gly Gly Val Trp Lys Gln Gly 115120 125 Trp Asn Ile Lys Tyr Asp Pro Leu Lys Tyr Asn Ala His His Lys Leu130 135 140 Lys Val Phe Val Val Pro His Ser His Asn Asp Pro Gly Trp IleGln 145 150 155 160 Thr Phe Glu Glu Tyr Tyr Gln His Asp Thr Lys His IleLeu Ser Asn 165 170 175 Ala Leu Arg His Leu His Asp Asn Pro Glu Met LysPhe Ile Trp Ala 180 185 190 Glu Ile Ser Tyr Phe Ala Arg Phe Tyr His AspLeu Gly Glu Asn Lys 195 200 205 Lys Leu Gln Met Lys Ser Ile Val Lys AsnGly Gln Leu Glu Phe Val 210 215 220 Thr Gly Gly Trp Val Met Pro Asp GluAla Asn Ser His Trp Arg Asn 225 230 235 240 Val Leu Leu Gln Leu Thr GluGly Gln Thr Trp Leu Lys Gln Phe Met 245 250 255 Asn Val Thr Pro Thr AlaSer Trp Ala Ile Asp Pro Phe Gly His Ser 260 265 270 Pro Thr Met Pro TyrIle Leu Gln Lys Ser Gly Phe Lys Asn Met Leu 275 280 285 Ile Gln Arg ThrHis Tyr Ser Val Lys Lys Glu Leu Ala Gln Gln Arg 290 295 300 Gln Leu GluPhe Leu Trp Arg Gln Ile Trp Asp Asn Lys Gly Asp Thr 305 310 315 320 AlaLeu Phe Thr His Met Met Pro Phe Tyr Ser Tyr Asp Ile Pro His 325 330 335Thr Cys Gly Pro Asp Pro Lys Val Cys Cys Gln Phe Asp Phe Lys Arg 340 345350 Met Gly Ser Phe Gly Leu Ser Cys Pro Trp Lys Val Pro Pro Arg Thr 355360 365 Ile Ser Asp Gln Asn Val Ala Ala Arg Ser Asp Leu Leu Val Asp Gln370 375 380 Trp Lys Lys Lys Ala Glu Leu Tyr Arg Thr Asn Val Leu Leu IlePro 385 390 395 400 Leu Gly Asp Asp Phe Arg Phe Lys Gln Asn Thr Glu TrpAsp Val Gln 405 410 415 Arg Val Asn Tyr Glu Arg Leu Phe Glu His Ile AsnSer Gln Ala His 420 425 430 Phe Asn Val Gln Ala Gln Phe Gly Thr Leu GlnGlu Tyr Phe Asp Ala 435 440 445 Val His Gln Ala Glu Arg Ala Gly Gln AlaGlu Phe Pro Thr Leu Ser 450 455 460 Gly Asp Phe Phe Thr Tyr Ala Asp ArgSer Asp Asn Tyr Trp Ser Gly 465 470 475 480 Tyr Tyr Thr Ser Arg Pro TyrHis Lys Arg Met Asp Arg Val Leu Met 485 490 495 His Tyr Val Arg Ala AlaGlu Met Leu Ser Ala Trp His Ser Trp Asp 500 505 510 Gly Met Ala Arg IleGlu Glu Arg Leu Glu Gln Ala Arg Arg Glu Leu 515 520 525 Ser Leu Phe GlnHis His Asp Gly Ile Thr Gly Thr Ala Lys Thr His 530 535 540 Val Val ValAsp Tyr Glu Gln Arg Met Gln Glu Ala Leu Lys Ala Cys 545 550 555 560 GlnMet Val Met Gln Gln Ser Val Tyr Arg Leu Leu Thr Lys Pro Ser 565 570 575Ile Tyr Ser Pro Asp Phe Ser Phe Ser Tyr Phe Thr Leu Asp Asp Ser 580 585590 Arg Trp Pro Gly Ser Gly Val Glu Asp Ser Arg Thr Thr Ile Ile Leu 595600 605 Gly Glu Asp Ile Leu Pro Ser Lys His Val Val Met His Asn Thr Leu610 615 620 Pro His Trp Arg Glu Gln Leu Val Asp Phe Tyr Val Ser Ser ProPhe 625 630 635 640 Val Ser Val Thr Asp Leu Ala Asn Asn Pro Val Glu AlaGln Val Ser 645 650 655 Pro Val Trp Ser Trp His His Asp Thr Leu Thr LysThr Ile His Pro 660 665 670 Gln Gly Ser Thr Thr Lys Tyr Arg Ile Ile PheLys Ala Arg Val Pro 675 680 685 Pro Met Gly Leu Ala Thr Tyr Val Leu ThrIle Ser Asp Ser Lys Pro 690 695 700 Glu His Thr Ser Tyr Ala Ser Asn LeuLeu Leu Arg Lys Asn Pro Thr 705 710 715 720 Ser Leu Pro Leu Gly Gln TyrPro Glu Asp Val Lys Phe Gly Asp Pro 725 730 735 Arg Glu Ile Ser Leu ArgVal Gly Asn Gly Pro Thr Leu Ala Phe Ser 740 745 750 Glu Gln Gly Leu LeuLys Ser Ile Gln Leu Thr Gln Asp Ser Pro His 755 760 765 Val Pro Val HisPhe Lys Phe Leu Lys Tyr Gly Val Arg Ser His Gly 770 775 780 Asp Arg SerGly Ala Tyr Leu Phe Leu Pro Asn Gly Pro Ala Ser Pro 785 790 795 800 ValGlu Leu Gly Gln Pro Val Val Leu Val Thr Lys Gly Lys Leu Glu 805 810 815Ser Ser Val Ser Val Gly Leu Pro Ser Val Val His Gln Thr Ile Met 820 825830 Arg Gly Gly Ala Pro Glu Ile Arg Asn Leu Val Asp Ile Gly Ser Leu 835840 845 Asp Asn Thr Glu Ile Val Met Arg Leu Glu Thr His Ile Asp Ser Gly850 855 860 Asp Ile Phe Tyr Thr Asp Leu Asn Gly Leu Gln Phe Ile Lys ArgArg 865 870 875 880 Arg Leu Asp Lys Leu Pro Leu Gln Ala Asn Tyr Tyr ProIle Pro Ser 885 890 895 Gly Met Phe Ile Glu Asp Ala Asn Thr Arg Leu ThrLeu Leu Thr Gly 900 905 910 Gln Pro Leu Gly Gly Ser Ser Leu Ala Ser GlyGlu Leu Glu Ile Met 915 920 925 Gln Asp Arg Arg Leu Ala Ser Asp Asp GluArg Gly Leu Gly Gln Gly 930 935 940 Val Leu Asp Asn Lys Pro Val Leu HisIle Tyr Arg Leu Val Leu Glu 945 950 955 960 Lys Val Asn Asn Cys Val ArgPro Ser Lys Leu His Pro Ala Gly Tyr 965 970 975 Leu Thr Ser Ala Ala HisLys Ala Ser Gln Ser Leu Leu Asp Pro Leu 980 985 990 Asp Lys Phe Ile PheAla Glu Asn Glu Trp Ile Gly Ala Gln Gly Gln 995 1000 1005 Phe Gly GlyAsp His Pro Ser Ala Arg Glu Asp Leu Asp Val Ser Val 1010 1015 1020 MetArg Arg Leu Thr Lys Ser Ser Ala Lys Thr Gln Arg Val Gly Tyr 1025 10301035 1040 Val Leu His Arg Thr Asn Leu Met Gln Cys Gly Thr Pro Glu GluHis 1045 1050 1055 Thr Gln Lys Leu Asp Val Cys His Leu Leu Pro Asn ValAla Arg Cys 1060 1065 1070 Glu Arg Thr Thr Leu Thr Phe Leu Gln Asn LeuGlu His Leu Asp Gly 1075 1080 1085 Met Val Ala Pro Glu Val Cys Pro MetGlu Thr Ala Ala Tyr Val Ser 1090 1095 1100 Ser His Ser Ser 1105 97 1144PRT Homo sapiens 97 Met Lys Leu Ser Arg Gln Phe Thr Val Phe Gly Ser AlaIle Phe Cys 1 5 10 15 Val Val Ile Phe Ser Leu Tyr Leu Met Leu Asp ArgGly His Leu Asp 20 25 30 Tyr Pro Arg Asn Pro Arg Arg Glu Gly Ser Phe ProGln Gly Gln Leu 35 40 45 Ser Met Leu Gln Glu Lys Ile Asp His Leu Glu ArgLeu Leu Ala Glu 50 55 60 Asn Asn Glu Ile Ile Ser Asn Ile Arg Asp Ser ValIle Asn Leu Ser 65 70 75 80 Glu Ser Val Glu Asp Gly Pro Lys Ser Ser GlnSer Asn Phe Ser Gln 85 90 95 Gly Ala Gly Ser His Leu Leu Pro Ser Gln LeuSer Leu Ser Val Asp 100 105 110 Thr Ala Asp Cys Leu Phe Ala Ser Gln SerGly Ser His Asn Ser Asp 115 120 125 Val Gln Met Leu Asp Val Tyr Ser LeuIle Ser Phe Asp Asn Pro Asp 130 135 140 Gly Gly Val Trp Lys Gln Gly PheAsp Ile Thr Tyr Glu Ser Asn Glu 145 150 155 160 Trp Asp Thr Glu Pro LeuGln Val Phe Val Val Pro His Ser His Asn 165 170 175 Asp Pro Gly Trp LeuLys Thr Phe Asn Asp Tyr Phe Arg Asp Lys Thr 180 185 190 Gln Tyr Ile PheAsn Asn Met Val Leu Lys Leu Lys Glu Asp Ser Arg 195 200 205 Arg Lys PheIle Trp Ser Glu Ile Ser Tyr Leu Ser Lys Trp Trp Asp 210 215 220 Ile IleAsp Ile Gln Lys Lys Asp Ala Val Lys Ser Leu Ile Glu Asn 225 230 235 240Gly Gln Leu Glu Ile Val Thr Gly Gly Trp Val Met Pro Asp Glu Ala 245 250255 Thr Pro His Tyr Phe Ala Leu Ile Asp Gln Leu Ile Glu Gly His Gln 260265 270 Trp Leu Glu Asn Asn Ile Gly Val Lys Pro Arg Ser Gly Trp Ala Ile275 280 285 Asp Pro Phe Gly His Ser Pro Thr Met Ala Tyr Leu Leu Asn ArgAla 290 295 300 Gly Leu Ser His Met Leu Ile Gln Arg Val His Tyr Ala ValLys Lys 305 310 315 320 His Phe Ala Leu His Lys Thr Leu Glu Phe Phe TrpArg Gln Asn Trp 325 330 335 Asp Leu Gly Ser Val Thr Asp Ile Leu Cys HisMet Met Pro Phe Tyr 340 345 350 Ser Tyr Asp Ile Pro His Thr Cys Gly ProAsp Pro Lys Ile Cys Cys 355 360 365 Gln Phe Asp Phe Lys Arg Leu Pro GlyGly Arg Phe Gly Cys Pro Trp 370 375 380 Gly Val Pro Pro Glu Thr Ile HisPro Gly Asn Val Gln Ser Arg Ala 385 390 395 400 Arg Met Leu Leu Asp GlnTyr Arg Lys Lys Ser Lys Leu Phe Arg Thr 405 410 415 Lys Val Leu Leu AlaPro Leu Gly Asp Asp Phe Arg Tyr Cys Glu Tyr 420 425 430 Thr Glu Trp AspLeu Gln Phe Lys Asn Tyr Gln Gln Leu Phe Asp Tyr 435 440 445 Met Asn SerGln Ser Lys Phe Lys Val Lys Ile Gln Phe Gly Thr Leu 450 455 460 Ser AspPhe Phe Asp Ala Leu Asp Lys Ala Asp Glu Thr Gln Arg Asp 465 470 475 480Lys Gly Gln Ser Met Phe Pro Val Leu Ser Gly Asp Phe Phe Thr Tyr 485 490495 Ala Asp Arg Asp Asp His Tyr Trp Ser Gly Tyr Phe Thr Ser Arg Pro 500505 510 Phe Tyr Lys Arg Met Asp Arg Ile Met Glu Ser His Leu Arg Ala Ala515 520 525 Glu Ile Leu Tyr Tyr Phe Ala Leu Arg Gln Ala His Lys Tyr LysIle 530 535 540 Asn Lys Phe Leu Ser Ser Ser Leu Tyr Thr Ala Leu Thr GluAla Arg 545 550 555 560 Arg Asn Leu Gly Leu Phe Gln His His Asp Ala IleThr Gly Thr Ala 565 570 575 Lys Asp Trp Val Val Val Asp Tyr Gly Thr ArgLeu Phe His Ser Leu 580 585 590 Met Val Leu Glu Lys Ile Ile Gly Asn SerAla Phe Leu Leu Ile Gly 595 600 605 Lys Asp Lys Leu Thr Tyr Asp Ser TyrSer Pro Asp Thr Phe Leu Glu 610 615 620 Met Asp Leu Lys Gln Lys Ser GlnAsp Ser Leu Pro Gln Lys Asn Ile 625 630 635 640 Ile Arg Leu Ser Ala GluPro Arg Tyr Leu Val Val Tyr Asn Pro Leu 645 650 655 Glu Gln Asp Arg IleSer Leu Val Ser Val Tyr Val Ser Ser Pro Thr 660 665 670 Val Gln Val PheSer Ala Ser Gly Lys Pro Val Glu Val Gln Val Ser 675 680 685 Ala Val TrpAsp Thr Ala Asn Thr Ile Ser Glu Thr Ala Tyr Glu Ile 690 695 700 Ser PheArg Ala His Ile Pro Pro Leu Gly Leu Lys Val Tyr Lys Ile 705 710 715 720Leu Glu Ser Ala Ser Ser Asn Ser His Leu Ala Asp Tyr Val Leu Tyr 725 730735 Lys Asn Lys Val Glu Asp Ser Gly Ile Phe Thr Ile Lys Asn Met Ile 740745 750 Asn Thr Glu Glu Gly Ile Thr Leu Glu Asn Ser Phe Val Leu Leu Arg755 760 765 Phe Asp Gln Thr Gly Leu Met Lys Gln Met Met Thr Lys Glu AspGly 770 775 780 Lys His His Glu Val Asn Val Gln Phe Ser Trp Tyr Gly ThrThr Ile 785 790 795 800 Lys Arg Asp Lys Ser Gly Ala Tyr Leu Phe Leu ProAsp Gly Asn Ala 805 810 815 Lys Pro Tyr Val Tyr Thr Thr Pro Pro Phe ValArg Val Thr His Gly 820 825 830 Arg Ile Tyr Ser Glu Val Thr Cys Phe PheAsp His Val Thr His Arg 835 840 845 Val Arg Leu Tyr His Ile Gln Gly IleGlu Gly Gln Ser Val Glu Val 850 855 860 Ser Asn Ile Val Asp Ile Arg LysVal Tyr Asn Arg Glu Ile Ala Met 865 870 875 880 Lys Ile Ser Ser Asp IleLys Ser Gln Asn Arg Phe Tyr Thr Asp Leu 885 890 895 Asn Gly Tyr Gln IleGln Pro Arg Met Thr Leu Ser Lys Leu Pro Leu 900 905 910 Gln Ala Asn ValTyr Pro Met Thr Thr Met Ala Tyr Ile Gln Asp Ala 915 920 925 Lys His ArgLeu Thr Leu Leu Ser Ala Gln Ser Leu Gly Val Ser Ser 930 935 940 Leu AsnSer Gly Gln Ile Glu Val Ile Met Asp Arg Arg Leu Met Gln 945 950 955 960Asp Asp Asn Arg Gly Leu Glu Gln Gly Ile Gln Asp Asn Lys Ile Thr 965 970975 Ala Asn Leu Phe Arg Ile Leu Leu Glu Lys Arg Ser Ala Val Asn Thr 980985 990 Glu Glu Glu Lys Lys Ser Val Ser Tyr Pro Ser Leu Leu Ser His Ile995 1000 1005 Thr Ser Ser Leu Met Asn His Pro Val Ile Pro Met Ala AsnLys Phe 1010 1015 1020 Ser Ser Pro Thr Leu Glu Leu Gln Gly Glu Phe SerPro Leu Gln Ser 1025 1030 1035 1040 Ser Leu Pro Cys Asp Ile His Leu ValAsn Leu Arg Thr Ile Gln Ser 1045 1050 1055 Lys Val Gly Asn Gly His SerAsn Glu Ala Ala Leu Ile Leu His Arg 1060 1065 1070 Lys Gly Phe Asp CysArg Phe Ser Ser Lys Gly Thr Gly Leu Phe Cys 1075 1080 1085 Ser Thr ThrGln Gly Lys Ile Leu Val Gln Lys Leu Leu Asn Lys Phe 1090 1095 1100 IleVal Glu Ser Leu Thr Pro Ser Ser Leu Ser Leu Met His Ser Pro 1105 11101115 1120 Pro Gly Thr Gln Asn Ile Ser Glu Ile Asn Leu Ser Pro Met GluIle 1125 1130 1135 Ser Thr Phe Arg Ile Gln Leu Arg 1140 98 1150 PRT Musmusculus 98 Met Lys Leu Ser Arg Gln Phe Thr Val Phe Gly Ser Ala Ile PheCys 1 5 10 15 Val Val Ile Phe Ser Leu Tyr Leu Met Leu Asp Arg Gly HisLeu Asp 20 25 30 Tyr Pro Arg Gly Pro Arg Gln Glu Gly Ser Phe Pro Gln GlyGln Leu 35 40 45 Ser Ile Leu Gln Glu Lys Ile Asp His Leu Glu Arg Leu LeuAla Glu 50 55 60 Asn Asn Glu Ile Ile Ser Asn Ile Arg Asp Ser Val Ile AsnLeu Ser 65 70 75 80 Glu Ser Val Glu Asp Gly Pro Arg Gly Ser Pro Gly AsnAla Ser Gln 85 90 95 Gly Ser Ile His Leu His Ser Pro Gln Leu Ala Leu GlnAla Asp Pro 100 105 110 Arg Asp Cys Leu Phe Ala Ser Gln Ser Gly Ser GlnPro Arg Asp Val 115 120 125 Gln Met Leu Asp Val Tyr Asp Leu Ile Pro PheAsp Asn Pro Asp Gly 130 135 140 Gly Val Trp Lys Gln Gly Phe Asp Ile LysTyr Glu Ala Asp Glu Trp 145 150 155 160 Asp His Glu Pro Leu Gln Val PheVal Val Pro His Ser His Asn Asp 165 170 175 Pro Gly Trp Leu Lys Thr PheAsn Asp Tyr Phe Arg Asp Lys Thr Gln 180 185 190 Tyr Ile Phe Asn Asn MetVal Leu Lys Leu Lys Glu Asp Ser Ser Arg 195 200 205 Lys Phe Met Trp SerGlu Ile Ser Tyr Leu Ala Lys Trp Trp Asp Ile 210 215 220 Ile Asp Ile ProLys Lys Glu Ala Val Lys Ser Leu Leu Gln Asn Gly 225 230 235 240 Gln LeuGlu Ile Val Thr Gly Gly Trp Val Met Pro Asp Glu Ala Thr 245 250 255 ProHis Tyr Phe Ala Leu Ile Asp Gln Leu Ile Glu Gly His Gln Trp 260 265 270Leu Glu Lys Asn Leu Gly Val Lys Pro Arg Ser Gly Trp Ala Ile Asp 275 280285 Pro Phe Gly His Ser Pro Thr Met Ala Tyr Leu Leu Lys Arg Ala Gly 290295 300 Phe Ser His Met Leu Ile Gln Arg Val His Tyr Ala Ile Lys Lys His305 310 315 320 Phe Ser Leu His Lys Thr Leu Glu Phe Phe Trp Arg Gln AsnTrp Asp 325 330 335 Leu Gly Ser Ala Thr Asp Ile Leu Cys His Met Met ProPhe Tyr Ser 340 345 350 Tyr Asp Ile Pro His Thr Cys Gly Pro Asp Pro LysIle Cys Cys Gln 355 360 365 Phe Asp Phe Lys Arg Leu Pro Gly Gly Arg TyrGly Cys Pro Trp Gly 370 375 380 Val Pro Pro Glu Ala Ile Ser Pro Gly AsnVal Gln Ser Arg Ala Gln 385 390 395 400 Met Leu Leu Asp Gln Tyr Arg LysLys Ser Lys Leu Phe Arg Thr Lys 405 410 415 Val Leu Leu Ala Pro Leu GlyAsp Asp Phe Arg Phe Ser Glu Tyr Thr 420 425 430 Glu Trp Asp Leu Gln CysArg Asn Tyr Glu Gln Leu Phe Ser Tyr Met 435 440 445 Asn Ser Gln Pro HisLeu Lys Val Lys Ile Gln Phe Gly Thr Leu Ser 450 455 460 Asp Tyr Phe AspAla Leu Glu Lys Ala Val Ala Ala Glu Lys Lys Ser 465 470 475 480 Ser GlnSer Val Phe Pro Ala Leu Ser Gly Asp Phe Phe Thr Tyr Ala 485 490 495 AspArg Asp Asp His Tyr Trp Ser Gly Tyr Phe Thr Ser Arg Pro Phe 500 505 510Tyr Lys Arg Met Asp Arg Ile Met Glu Ser Arg Ile Arg Ala Ala Glu 515 520525 Ile Leu Tyr Gln Leu Ala Leu Lys Gln Ala Gln Lys Tyr Lys Ile Asn 530535 540 Lys Phe Leu Ser Ser Pro His Tyr Thr Thr Leu Thr Glu Ala Arg Arg545 550 555 560 Asn Leu Gly Leu Phe Gln His His Asp Ala Ile Thr Gly ThrAla Lys 565 570 575 Asp Trp Val Val Val Asp Tyr Gly Thr Arg Leu Phe GlnSer Leu Asn 580 585 590 Ser Leu Glu Lys Ile Ile Gly Asp Ser Ala Phe LeuLeu Ile Leu Lys 595 600 605 Asp Lys Lys Leu Tyr Gln Ser Asp Pro Ser LysAla Phe Leu Glu Met 610 615 620 Asp Thr Lys Gln Ser Ser Gln Asp Ser LeuPro Gln Lys Ile Ile Ile 625 630 635 640 Gln Leu Ser Ala Gln Glu Pro ArgTyr Leu Val Val Tyr Asn Pro Phe 645 650 655 Glu Gln Glu Arg His Ser ValVal Ser Ile Arg Val Asn Ser Ala Thr 660 665 670 Gly Lys Val Leu Ser AspSer Gly Lys Pro Val Glu Val Gln Val Ser 675 680 685 Ala Val Trp Asn AspMet Arg Thr Ile Ser Gln Ala Ala Tyr Glu Val 690 695 700 Ser Phe Leu AlaHis Ile Pro Pro Leu Gly Leu Lys Val Phe Lys Ile 705 710 715 720 Leu GluSer Gln Ser Ser Ser Ser His Leu Ala Asp Tyr Val Leu Tyr 725 730 735 AsnAsn Asp Gly Leu Ala Glu Asn Gly Ile Phe His Val Lys Asn Met 740 745 750Val Asp Ala Gly Asp Ala Ile Thr Ile Glu Asn Pro Phe Leu Ala Ile 755 760765 Trp Phe Asp Arg Ser Gly Leu Met Glu Lys Val Arg Arg Lys Glu Asp 770775 780 Ser Arg Gln His Glu Leu Lys Val Gln Phe Leu Trp Tyr Gly Thr Thr785 790 795 800 Asn Lys Arg Asp Lys Ser Gly Ala Tyr Leu Phe Leu Pro AspGly Gln 805 810 815 Gly Gln Pro Tyr Val Ser Leu Arg Pro Pro Phe Val ArgVal Thr Arg 820 825 830 Gly Arg Ile Tyr Ser Asp Val Thr Cys Phe Leu GluHis Val Thr His 835 840 845 Lys Val Arg Leu Tyr Asn Ile Gln Gly Ile GluGly Gln Ser Met Glu 850 855 860 Val Ser Asn Ile Val Asn Ile Arg Asn ValHis Asn Arg Glu Ile Val 865 870 875 880 Met Arg Ile Ser Ser Lys Ile AsnAsn Gln Asn Arg Tyr Tyr Thr Asp 885 890 895 Leu Asn Gly Tyr Gln Ile GlnPro Arg Arg Thr Met Ser Lys Leu Pro 900 905 910 Leu Gln Ala Asn Val TyrPro Met Cys Thr Met Ala Tyr Ile Gln Asp 915 920 925 Ala Glu His Arg LeuThr Leu Leu Ser Ala Gln Ser Leu Gly Ala Ser 930 935 940 Ser Met Ala SerGly Gln Ile Glu Val Phe Met Asp Arg Arg Leu Met 945 950 955 960 Gln AspAsp Asn Arg Gly Leu Gly Gln Gly Val His Asp Asn Lys Ile 965 970 975 ThrAla Asn Leu Phe Arg Ile Leu Leu Glu Lys Arg Ser Ala Val Asn 980 985 990Met Glu Glu Glu Lys Lys Ser Pro Val Ser Tyr Pro Ser Leu Leu Ser 995 10001005 His Met Thr Ser Ser Phe Leu Asn His Pro Phe Leu Pro Met Val Leu1010 1015 1020 Ser Gly Gln Leu Pro Ser Pro Ala Phe Glu Leu Leu Ser GluPhe Pro 1025 1030 1035 1040 Leu Leu Gln Ser Ser Leu Pro Cys Asp Ile HisLeu Val Asn Leu Arg 1045 1050 1055 Thr Ile Gln Ser Lys Met Gly Lys GlyTyr Ser Asp Glu Ala Ala Leu 1060 1065 1070 Ile Leu His Arg Lys Gly PheAsp Cys Gln Phe Ser Ser Arg Gly Ile 1075 1080 1085 Gly Leu Pro Cys SerThr Thr Gln Gly Lys Met Ser Val Leu Lys Leu 1090 1095 1100 Phe Asn LysPhe Ala Val Glu Ser Leu Val Pro Ser Ser Leu Ser Leu 1105 1110 1115 1120Met His Ser Pro Pro Asp Ala Gln Asn Met Ser Glu Val Ser Leu Ser 11251130 1135 Pro Met Glu Ile Ser Thr Phe Arg Ile Arg Leu Arg Trp Thr 11401145 1150 99 1139 PRT Homo sapiens 99 Met Lys Leu Lys Lys Gln Val ThrVal Cys Gly Ala Ala Ile Phe Cys 1 5 10 15 Val Ala Val Phe Ser Leu TyrLeu Met Leu Asp Arg Val Gln His Asp 20 25 30 Pro Thr Arg His Gln Asn GlyGly Asn Phe Pro Arg Ser Gln Ile Ser 35 40 45 Val Leu Gln Asn Arg Ile GluGln Leu Glu Gln Leu Leu Glu Glu Asn 50 55 60 His Glu Ile Ile Ser His IleLys Asp Ser Val Leu Glu Leu Thr Ala 65 70 75 80 Asn Ala Glu Gly Pro ProAla Met Leu Pro Tyr Tyr Thr Val Asn Gly 85 90 95 Ser Trp Val Val Pro ProGlu Pro Arg Pro Ser Phe Phe Ser Ile Ser 100 105 110 Pro Gln Asp Cys GlnPhe Ala Leu Gly Gly Arg Gly Gln Lys Pro Glu 115 120 125 Leu Gln Met LeuThr Val Ser Glu Glu Leu Pro Phe Asp Asn Val Asp 130 135 140 Gly Gly ValTrp Arg Gln Gly Phe Asp Ile Ser Tyr Asp Pro His Asp 145 150 155 160 TrpAsp Ala Glu Asp Leu Gln Val Phe Val Val Pro His Ser His Asn 165 170 175Asp Pro Gly Trp Ile Lys Thr Phe Asp Lys Tyr Tyr Thr Glu Gln Thr 180 185190 Gln His Ile Leu Asn Ser Met Val Ser Lys Leu Gln Glu Asp Pro Arg 195200 205 Arg Arg Phe Leu Trp Ala Glu Val Ser Phe Phe Ala Lys Trp Trp Asp210 215 220 Asn Ile Asn Val Gln Lys Arg Ala Ala Val Arg Arg Leu Val GlyAsn 225 230 235 240 Gly Gln Leu Glu Ile Ala Thr Gly Gly Trp Val Met ProAsp Glu Ala 245 250 255 Asn Ser His Tyr Phe Ala Leu Ile Asp Gln Leu IleGlu Gly His Gln 260 265 270 Trp Leu Glu Arg Asn Leu Gly Ala Thr Pro ArgSer Gly Trp Ala Val 275 280 285 Asp Pro Phe Gly Tyr Ser Ser Thr Met ProTyr Leu Leu Arg Arg Ala 290 295 300 Asn Leu Thr Ser Met Leu Ile Gln ArgVal His Tyr Ala Ile Lys Lys 305 310 315 320 His Phe Ala Ala Thr His SerLeu Glu Phe Met Trp Arg Gln Thr Trp 325 330 335 Asp Ser Asp Ser Ser ThrAsp Ile Phe Cys His Met Met Pro Phe Tyr 340 345 350 Ser Tyr Asp Val ProHis Thr Cys Gly Pro Asp Pro Lys Ile Cys Cys 355 360 365 Gln Phe Asp PheLys Arg Leu Pro Gly Gly Arg Ile Asn Cys Pro Trp 370 375 380 Lys Val ProPro Arg Ala Ile Thr Glu Ala Asn Val Ala Glu Arg Ala 385 390 395 400 AlaLeu Leu Leu Asp Gln Tyr Arg Lys Lys Ser Gln Leu Phe Arg Ser 405 410 415Asn Val Leu Leu Val Pro Leu Gly Asp Asp Phe Arg Tyr Asp Lys Pro 420 425430 Gln Glu Trp Asp Ala Gln Phe Phe Asn Tyr Gln Arg Leu Phe Asp Phe 435440 445 Phe Asn Ser Arg Pro Asn Leu His Val Gln Ala Gln Phe Gly Thr Leu450 455 460 Ser Asp Tyr Phe Asp Ala Leu Tyr Lys Arg Thr Gly Val Glu ProGly 465 470 475 480 Ala Arg Pro Pro Gly Phe Pro Val Leu Ser Gly Asp PhePhe Ser Tyr 485 490 495 Ala Asp Arg Glu Asp His Tyr Trp Thr Gly Tyr TyrThr Ser Arg Pro 500 505 510 Phe Tyr Lys Ser Leu Asp Arg Val Leu Glu AlaHis Leu Arg Gly Ala 515 520 525 Glu Val Leu Tyr Ser Leu Ala Ala Ala HisAla Arg Arg Ser Gly Leu 530 535 540 Ala Gly Arg Tyr Pro Leu Ser Asp PheThr Leu Leu Thr Glu Ala Arg 545 550 555 560 Arg Thr Leu Gly Leu Phe GlnHis His Asp Ala Ile Thr Gly Thr Ala 565 570 575 Lys Glu Ala Val Val ValAsp Tyr Gly Val Arg Leu Leu Arg Ser Leu 580 585 590 Val Asn Leu Lys GlnVal Ile Ile His Ala Ala His Tyr Leu Val Leu 595 600 605 Gly Asp Lys GluThr Tyr His Phe Asp Pro Glu Ala Pro Phe Leu Gln 610 615 620 Val Asp AspThr Arg Leu Ser His Asp Ala Leu Pro Glu Arg Thr Val 625 630 635 640 IleGln Leu Asp Ser Ser Pro Arg Phe Val Val Leu Phe Asn Pro Leu 645 650 655Glu Gln Glu Arg Phe Ser Met Val Ser Leu Leu Val Asn Ser Pro Arg 660 665670 Val Arg Val Leu Ser Glu Glu Gly Gln Pro Leu Ala Val Gln Ile Ser 675680 685 Ala His Trp Ser Ser Ala Thr Glu Ala Val Pro Asp Val Tyr Gln Val690 695 700 Ser Val Pro Val Arg Leu Pro Ala Leu Gly Leu Gly Val Leu GlnLeu 705 710 715 720 Gln Leu Gly Leu Asp Gly His Arg Thr Leu Pro Ser SerVal Arg Ile 725 730 735 Tyr Leu His Gly Arg Gln Leu Ser Val Ser Arg HisGlu Ala Phe Pro 740 745 750 Leu Arg Val Ile Asp Ser Gly Thr Ser Asp PheAla Leu Ser Asn Arg 755 760 765 Tyr Met Gln Val Trp Phe Ser Gly Leu ThrGly Leu Leu Lys Ser Ile 770 775 780 Arg Arg Val Asp Glu Glu His Glu GlnGln Val Asp Met Gln Val Leu 785 790 795 800 Val Tyr Gly Thr Arg Thr SerLys Asp Lys Ser Gly Ala Tyr Leu Phe 805 810 815 Leu Pro Asp Gly Glu AlaSer Pro Thr Ser Pro Arg Ser Pro Pro Cys 820 825 830 Cys Val Ser Leu LysAla Leu Ser Ser Gln Arg Trp Leu Arg Thr Met 835 840 845 Ser Thr Phe ThrArg Arg Ser Gly Phe Thr Ile Cys Gln Gly Trp Arg 850 855 860 Gly Cys LeuTrp Thr Tyr His Pro Trp Trp Thr Ser Gly Thr Thr Ser 865 870 875 880 ThrArg Ser Trp Pro Cys Thr Ser Ile Gln Thr Ser Thr Ala Arg Val 885 890 895Gln Pro Arg Arg Tyr Leu Lys Lys Leu Pro Leu Gln Ala Asn Phe Tyr 900 905910 Pro Met Pro Val Met Ala Tyr Ile Gln Asp Ala Gln Lys Arg Leu Thr 915920 925 Leu His Thr Ala Gln Ala Leu Gly Val Ser Ser Leu Lys Asp Gly Gln930 935 940 Leu Glu Val Ile Leu Asp Arg Arg Leu Met Gln Asp Asp Asn ArgGly 945 950 955 960 Leu Gly Gln Gly Leu Lys Asp Asn Lys Arg Thr Cys AsnArg Phe Arg 965 970 975 Leu Leu Leu Glu Arg Arg Thr Val Gly Ser Glu ValGln Asp Ser His 980 985 990 Ser Thr Ser Tyr Pro Ser Leu Leu Ser His LeuThr Ser Met Tyr Leu 995 1000 1005 Asn Ala Pro Ala Leu Ala Leu Pro ValAla Arg Met Gln Leu Pro Gly 1010 1015 1020 Pro Gly Leu Arg Ser Phe HisPro Leu Ala Ser Ser Leu Pro Cys Asp 1025 1030 1035 1040 Phe His Leu LeuAsn Leu Arg Thr Leu Gln Ala Glu Glu Asp Thr Leu 1045 1050 1055 Pro SerAla Glu Thr Ala Leu Ile Leu His Arg Lys Gly Phe Asp Cys 1060 1065 1070Gly Leu Glu Ala Lys Asn Leu Gly Phe Asn Cys Thr Thr Ser Gln Gly 10751080 1085 Lys Val Ala Leu Gly Ser Leu Phe His Gly Leu Asp Val Val PheLeu 1090 1095 1100 Gln Pro Thr Ser Leu Thr Leu Leu Tyr Pro Leu Ala SerPro Ser Asn 1105 1110 1115 1120 Ser Thr Asp Val Tyr Leu Glu Pro Met GluIle Ala Thr Phe Arg Leu 1125 1130 1135 Arg Leu Gly 100 1130 PRTSpodoptera frugiperda 100 Met Arg Thr Arg Val Leu Arg Cys Arg Pro PheSer Thr Arg Ile Leu 1 5 10 15 Leu Leu Leu Leu Phe Val Leu Ala Phe GlyVal Tyr Cys Tyr Phe Tyr 20 25 30 Asn Ala Ser Pro Gln Asn Tyr Asn Lys ProArg Ile Ser Tyr Pro Ala 35 40 45 Ser Met Glu His Phe Lys Ser Ser Leu ThrHis Thr Val Lys Ser Arg 50 55 60 Asp Glu Pro Thr Pro Asp Gln Cys Pro AlaLeu Lys Glu Ser Glu Ala 65 70 75 80 Asp Ile Asp Thr Val Ala Ile Tyr ProThr Phe Asp Phe Gln Pro Ser 85 90 95 Trp Leu Arg Thr Lys Glu Phe Trp AspLys Ser Phe Glu Asp Arg Tyr 100 105 110 Glu Arg Ile His Asn Asp Thr ThrArg Pro Arg Leu Lys Val Ile Val 115 120 125 Val Pro His Ser His Asn AspPro Gly Trp Leu Lys Thr Phe Glu Gln 130 135 140 Tyr Phe Glu Trp Lys ThrLys Asn Ile Ile Asn Asn Ile Val Asn Lys 145 150 155 160 Leu His Gln TyrPro Asn Met Thr Phe Ile Trp Thr Glu Ile Ser Phe 165 170 175 Leu Asn AlaTrp Trp Glu Arg Ser His Pro Val Lys Gln Lys Ala Leu 180 185 190 Lys LysLeu Ile Lys Glu Gly Arg Leu Glu Ile Thr Thr Gly Gly Trp 195 200 205 ValMet Pro Asp Glu Ala Cys Thr His Ile Tyr Ala Leu Ile Asp Gln 210 215 220Phe Ile Glu Gly His His Trp Val Lys Thr Asn Leu Gly Val Ile Pro 225 230235 240 Lys Thr Gly Trp Ser Ile Asp Pro Phe Gly His Gly Ala Thr Val Pro245 250 255 Tyr Leu Leu Asp Gln Ser Gly Leu Glu Gly Thr Ile Ile Gln ArgIle 260 265 270 His Tyr Ala Trp Lys Gln Trp Leu Ala Glu Arg Gln Ile GluGlu Phe 275 280 285 Tyr Trp Leu Ala Ser Trp Ala Thr Thr Lys Pro Ser MetIle Val His 290 295 300 Asn Gln Pro Phe Asp Ile Tyr Ser Ile Lys Ser ThrCys Gly Pro His 305 310 315 320 Pro Ser Ile Cys Leu Ser Phe Asp Phe ArgLys Ile Pro Gly Glu Tyr 325 330 335 Ser Glu Tyr Thr Ala Lys His Glu AspIle Thr Glu His Asn Leu His 340 345 350 Ser Lys Ala Lys Thr Leu Ile GluGlu Tyr Asp Arg Ile Gly Ser Leu 355 360 365 Thr Pro His Asn Val Val LeuVal Pro Leu Gly Asp Asp Phe Arg Tyr 370 375 380 Glu Tyr Ser Val Glu PheAsp Ala Gln Tyr Val Asn Tyr Met Lys Met 385 390 395 400 Phe Asn Tyr IleAsn Ala His Lys Glu Ile Phe Asn Ala Asp Val Gln 405 410 415 Phe Gly ThrPro Leu Asp Tyr Phe Asn Ala Met Lys Glu Arg His Gln 420 425 430 Asn IlePro Ser Leu Lys Gly Asp Phe Phe Val Tyr Ser Asp Ile Phe 435 440 445 SerGlu Gly Lys Pro Ala Tyr Trp Ser Gly Tyr Tyr Thr Thr Arg Pro 450 455 460Tyr Gln Lys Ile Leu Ala Arg Gln Phe Glu His Gln Leu Arg Ser Ala 465 470475 480 Glu Ile Leu Phe Thr Leu Val Ser Asn Tyr Ile Arg Gln Met Gly Arg485 490 495 Gln Gly Glu Phe Gly Ala Ser Glu Lys Lys Leu Glu Lys Ser TyrGlu 500 505 510 Gln Leu Ile Tyr Ala Arg Arg Asn Leu Gly Leu Phe Gln HisHis Asp 515 520 525 Ala Ile Thr Gly Thr Ser Lys Ser Ser Val Met Gln AspTyr Gly Thr 530 535 540 Lys Leu Phe Thr Ser Leu Tyr His Cys Ile Arg LeuGln Glu Ala Ala 545 550 555 560 Leu Thr Thr Ile Met Leu Pro Asp Gln SerLeu His Ser Gln Ser Ile 565 570 575 Ile Gln Ser Glu Val Glu Trp Glu ThrTyr Gly Lys Pro Pro Lys Lys 580 585 590 Leu Gln Val Ser Phe Ile Asp LysLys Lys Val Ile Leu Phe Asn Pro 595 600 605 Leu Ala Glu Thr Arg Thr GluVal Val Thr Val Arg Ser Asn Thr Ser 610 615 620 Asn Ile Arg Val Tyr AspThr His Lys Arg Lys His Val Leu Tyr Gln 625 630 635 640 Ile Met Pro SerIle Thr Ile Gln Asp Asn Gly Lys Ser Ile Val Ser 645 650 655 Asp Thr ThrPhe Asp Ile Met Phe Val Ala Thr Ile Pro Pro Leu Thr 660 665 670 Ser IleSer Tyr Lys Leu Gln Glu His Thr Asn Thr Ser His His Cys 675 680 685 ValIle Phe Cys Asn Asn Cys Glu Gln Tyr Gln Lys Ser Asn Val Phe 690 695 700Gln Ile Lys Lys Met Met Pro Gly Asp Ile Gln Leu Glu Asn Ala Val 705 710715 720 Leu Lys Leu Leu Val Asn Arg Asn Thr Gly Phe Leu Arg Gln Val Tyr725 730 735 Arg Lys Asp Ile Arg Lys Arg Thr Val Val Asp Val Gln Phe GlyAla 740 745 750 Tyr Gln Ser Ala Gln Arg His Ser Gly Ala Tyr Leu Phe MetPro His 755 760 765 Tyr Asp Ser Pro Glu Lys Asn Val Leu His Pro Tyr ThrAsn Gln Asn 770 775 780 Asn Met Gln Asp Asp Asn Ile Ile Ile Val Ser GlyPro Ile Ser Thr 785 790 795 800 Glu Ile Thr Thr Met Tyr Leu Pro Phe LeuVal His Thr Ile Arg Ile 805 810 815 Tyr Asn Val Pro Asp Pro Val Leu SerArg Ala Ile Leu Leu Glu Thr 820 825 830 Asp Val Asp Phe Glu Ala Pro ProLys Asn Arg Glu Thr Glu Leu Phe 835 840 845 Met Arg Leu Gln Thr Asp IleGln Asn Gly Asp Ile Pro Glu Phe Tyr 850 855 860 Thr Asp Gln Asn Gly PheGln Tyr Gln Lys Arg Val Lys Val Asn Lys 865 870 875 880 Leu Gly Ile GluAla Asn Tyr Tyr Pro Ile Thr Thr Met Ala Cys Leu 885 890 895 Gln Asp GluGlu Thr Arg Leu Thr Leu Leu Thr Asn His Ala Gln Gly 900 905 910 Ala AlaAla Tyr Glu Pro Gly Arg Leu Glu Val Met Leu Asp Arg Arg 915 920 925 ThrLeu Tyr Asp Asp Phe Arg Gly Ile Gly Glu Gly Val Val Asp Asn 930 935 940Lys Pro Thr Thr Phe Gln Asn Trp Ile Leu Ile Glu Ser Met Pro Gly 945 950955 960 Val Thr Arg Ala Lys Arg Asp Thr Ser Glu Pro Gly Phe Lys Phe Val965 970 975 Asn Glu Arg Arg Phe Gly Pro Gly Gln Lys Glu Ser Pro Tyr GlnVal 980 985 990 Pro Ser Gln Thr Ala Asp Tyr Leu Ser Arg Met Phe Asn TyrPro Val 995 1000 1005 Asn Val Tyr Leu Val Asp Thr Ser Glu Val Gly GluIle Glu Val Lys 1010 1015 1020 Pro Tyr Gln Ser Phe Leu Gln Ser Phe ProPro Gly Ile His Leu Val 1025 1030 1035 1040 Thr Leu Arg Thr Ile Thr AspAsp Val Leu Glu Leu Phe Pro Ser Asn 1045 1050 1055 Glu Ser Tyr Met ValLeu His Arg Pro Gly Tyr Ser Cys Ala Val Gly 1060 1065 1070 Glu Lys ProVal Ala Lys Ser Pro Lys Phe Ser Ser Lys Thr Arg Phe 1075 1080 1085 AsnGly Leu Asn Ile Gln Asn Ile Thr Ala Val Ser Leu Thr Gly Leu 1090 10951100 Lys Ser Leu Arg Pro Leu Thr Gly Leu Ser Asp Ile His Leu Asn Ala1105 1110 1115 1120 Met Glu Val Lys Thr Tyr Lys Ile Arg Phe 1125 1130101 1010 PRT Homo sapiens 101 Met Gly Tyr Ala Arg Ala Ser Gly Val CysAla Arg Gly Cys Leu Asp 1 5 10 15 Ser Ala Gly Pro Trp Thr Met Ser ArgAla Leu Arg Pro Pro Leu Pro 20 25 30 Pro Leu Cys Phe Phe Leu Leu Leu LeuAla Ala Ala Gly Ala Arg Ala 35 40 45 Gly Gly Tyr Glu Thr Cys Pro Thr ValGln Pro Asn Met Leu Asn Val 50 55 60 His Leu Leu Pro His Thr His Asp AspVal Gly Trp Leu Lys Thr Val 65 70 75 80 Asp Gln Tyr Phe Tyr Gly Ile LysAsn Asp Ile Gln His Ala Gly Val 85 90 95 Gln Tyr Ile Leu Asp Ser Val IleSer Ala Leu Leu Ala Asp Pro Thr 100 105 110 Arg Arg Phe Ile Tyr Val GluIle Ala Phe Phe Ser Arg Trp Trp His 115 120 125 Gln Gln Thr Asn Ala ThrGln Glu Val Val Arg Asp Leu Val Arg Gln 130 135 140 Gly Arg Leu Glu PheAla Asn Gly Gly Trp Val Met Asn Asp Glu Ala 145 150 155 160 Ala Thr HisTyr Gly Ala Ile Val Asp Gln Met Thr Leu Gly Leu Arg 165 170 175 Phe LeuGlu Asp Thr Phe Gly Asn Asp Gly Arg Pro Arg Val Ala Trp 180 185 190 HisIle Asp Pro Phe Gly His Ser Arg Glu Gln Ala Ser Leu Phe Ala 195 200 205Gln Met Gly Phe Asp Gly Phe Phe Phe Gly Arg Leu Asp Tyr Gln Asp 210 215220 Lys Trp Val Arg Met Gln Lys Leu Glu Met Glu Gln Val Trp Arg Ala 225230 235 240 Ser Thr Ser Leu Lys Pro Pro Thr Ala Asp Leu Phe Thr Gly ValLeu 245 250 255 Pro Asn Gly Tyr Asn Pro Pro Arg Asn Leu Cys Trp Asp ValLeu Cys 260 265 270 Val Asp Gln Pro Leu Val Glu Asp Pro Arg Ser Pro GluTyr Asn Ala 275 280 285 Lys Glu Leu Val Asp Tyr Phe Leu Asn Val Ala ThrAla Gln Gly Arg 290 295 300 Tyr Tyr Arg Thr Asn His Thr Val Met Thr MetGly Ser Asp Phe Gln 305 310 315 320 Tyr Glu Asn Ala Asn Met Trp Phe LysAsn Leu Asp Lys Leu Ile Arg 325 330 335 Leu Val Asn Ala Gln Gln Ala LysGly Ser Ser Val His Val Leu Tyr 340 345 350 Ser Thr Pro Ala Cys Tyr LeuTrp Glu Leu Asn Lys Ala Asn Leu Thr 355 360 365 Trp Ser Val Lys His AspAsp Phe Phe Pro Tyr Ala Asp Gly Pro His 370 375 380 Gln Phe Trp Thr GlyTyr Phe Ser Ser Arg Pro Ala Leu Lys Arg Tyr 385 390 395 400 Glu Arg LeuSer Tyr Asn Phe Leu Gln Val Cys Asn Gln Leu Glu Ala 405 410 415 Leu ValGly Leu Ala Ala Asn Val Gly Pro Tyr Gly Ser Gly Asp Ser 420 425 430 AlaPro Leu Asn Glu Ala Met Ala Val Leu Gln His His Asp Ala Val 435 440 445Ser Gly Thr Ser Arg Gln His Val Ala Asn Asp Tyr Ala Arg Gln Leu 450 455460 Ala Ala Gly Trp Gly Pro Cys Glu Val Leu Leu Ser Asn Ala Leu Ala 465470 475 480 Arg Leu Arg Gly Phe Lys Asp His Phe Thr Phe Cys Gln Gln LeuAsn 485 490 495 Ile Ser Ile Cys Pro Leu Ser Gln Thr Ala Ala Arg Phe GlnVal Ile 500 505 510 Val Tyr Asn Pro Leu Gly Arg Lys Val Asn Trp Met ValArg Leu Pro 515 520 525 Val Ser Glu Gly Val Phe Val Val Lys Asp Pro AsnGly Arg Thr Val 530 535 540 Pro Ser Asp Val Val Ile Phe Pro Ser Ser AspSer Gln Ala His Pro 545 550 555 560 Pro Glu Leu Leu Phe Ser Ala Ser LeuPro Ala Leu Gly Phe Ser Thr 565 570 575 Tyr Ser Val Ala Gln Val Pro ArgTrp Lys Pro Gln Ala Arg Ala Pro 580 585 590 Gln Pro Ile Pro Arg Arg SerTrp Ser Pro Ala Leu Thr Ile Glu Asn 595 600 605 Glu His Ile Arg Ala ThrPhe Asp Pro Asp Thr Gly Leu Leu Met Glu 610 615 620 Ile Met Asn Met AsnGln Gln Leu Leu Leu Pro Val Arg Gln Thr Phe 625 630 635 640 Phe Trp TyrAsn Ala Ser Ile Gly Asp Asn Glu Ser Asp Gln Ala Ser 645 650 655 Gly AlaTyr Ile Phe Arg Pro Asn Gln Gln Lys Pro Leu Pro Val Ser 660 665 670 ArgTrp Ala Gln Ile His Leu Val Lys Thr Pro Leu Val Gln Glu Val 675 680 685His Gln Asn Phe Ser Ala Trp Cys Ser Gln Val Val Arg Leu Tyr Pro 690 695700 Gly Gln Arg His Leu Glu Leu Glu Trp Ser Val Gly Pro Ile Pro Val 705710 715 720 Gly Asp Thr Trp Gly Lys Glu Val Ile Ser Arg Phe Asp Thr ProLeu 725 730 735 Glu Thr Lys Gly Arg Phe Tyr Thr Asp Ser Asn Gly Arg GluIle Leu 740 745 750 Glu Arg Arg Arg Asp Tyr Arg Pro Thr Trp Lys Leu AsnGln Thr Glu 755 760 765 Pro Val Ala Gly Asn Tyr Tyr Pro Val Asn Thr ArgIle Tyr Ile Thr 770 775 780 Asp Gly Asn Met Gln Leu Thr Val Leu Thr AspArg Ser Gln Gly Gly 785 790 795 800 Ser Ser Leu Arg Asp Gly Ser Leu GluLeu Met Val His Arg Arg Leu 805 810 815 Leu Lys Asp Asp Gly Arg Gly ValSer Glu Pro Leu Met Glu Asn Gly 820 825 830 Ser Gly Ala Trp Val Arg GlyArg His Leu Val Leu Leu Asp Thr Ala 835 840 845 Gln Ala Ala Ala Ala GlyHis Arg Leu Leu Ala Glu Gln Glu Val Leu 850 855 860 Ala Pro Gln Val ValLeu Ala Pro Gly Gly Gly Ala Ala Tyr Asn Leu 865 870 875 880 Gly Ala ProPro Arg Thr Gln Phe Ser Gly Leu Arg Arg Asp Leu Pro 885 890 895 Pro SerVal His Leu Leu Thr Leu Ala Ser Trp Gly Pro Glu Met Val 900 905 910 LeuLeu Arg Leu Glu His Gln Phe Ala Val Gly Glu Asp Ser Gly Arg 915 920 925Asn Leu Ser Ala Pro Val Thr Leu Asn Leu Arg Asp Leu Phe Ser Thr 930 935940 Phe Thr Ile Thr Arg Leu Gln Glu Thr Thr Leu Val Ala Asn Gln Leu 945950 955 960 Arg Glu Ala Ala Ser Arg Leu Lys Trp Thr Thr Asn Thr Gly ProThr 965 970 975 Pro His Gln Thr Pro Tyr Gln Leu Asp Pro Ala Asn Ile ThrLeu Glu 980 985 990 Pro Met Glu Ile Arg Thr Phe Leu Ala Ser Val Gln TrpLys Glu Val 995 1000 1005 Asp Gly 1010 102 1062 PRT Homo sapiens 102 MetAla Ala Ala Pro Phe Leu Lys His Trp Arg Thr Thr Phe Glu Arg 1 5 10 15Val Glu Lys Phe Val Ser Pro Ile Tyr Phe Thr Asp Cys Asn Leu Arg 20 25 30Gly Arg Leu Phe Gly Ala Ser Cys Pro Val Ala Val Leu Ser Ser Phe 35 40 45Leu Thr Pro Glu Arg Leu Pro Tyr Gln Glu Ala Val Gln Arg Asp Phe 50 55 60Arg Pro Ala Gln Val Gly Asp Ser Phe Gly Pro Thr Trp Trp Thr Cys 65 70 7580 Trp Phe Arg Val Glu Leu Thr Ile Pro Glu Ala Trp Val Gly Gln Glu 85 9095 Val His Leu Cys Trp Glu Ser Asp Gly Glu Gly Leu Val Trp Arg Asp 100105 110 Gly Glu Pro Val Gln Gly Leu Thr Lys Glu Gly Glu Lys Thr Ser Tyr115 120 125 Val Leu Thr Asp Arg Leu Gly Glu Arg Asp Pro Arg Ser Leu ThrLeu 130 135 140 Tyr Val Glu Val Ala Cys Asn Gly Leu Leu Gly Ala Gly LysGly Ser 145 150 155 160 Met Ile Ala Ala Pro Asp Pro Glu Lys Ile Phe GlnLeu Ser Arg Ala 165 170 175 Glu Leu Ala Val Phe His Arg Asp Val His MetLeu Leu Val Asp Leu 180 185 190 Glu Leu Leu Leu Gly Ile Ala Lys Gly LeuGly Lys Asp Asn Gln Arg 195 200 205 Ser Phe Gln Ala Leu Tyr Thr Ala AsnGln Met Val Asn Val Cys Asp 210 215 220 Pro Ala Gln Pro Glu Thr Phe ProVal Ala Gln Ala Leu Ala Ser Arg 225 230 235 240 Phe Phe Gly Gln His GlyGly Glu Ser Gln His Thr Ile His Ala Thr 245 250 255 Gly His Cys His IleAsp Thr Ala Trp Leu Trp Pro Phe Lys Glu Thr 260 265 270 Val Arg Lys CysAla Arg Ser Trp Val Thr Ala Leu Gln Leu Met Glu 275 280 285 Arg Asn ProGlu Phe Ile Phe Ala Cys Ser Gln Ala Gln Gln Leu Glu 290 295 300 Trp ValLys Ser Arg Tyr Pro Gly Leu Tyr Ser Arg Ile Gln Glu Phe 305 310 315 320Ala Cys Arg Gly Gln Phe Val Pro Val Gly Gly Thr Trp Val Glu Met 325 330335 Asp Gly Asn Leu Pro Ser Gly Glu Ala Met Val Arg Gln Phe Leu Gln 340345 350 Gly Gln Asn Phe Phe Leu Gln Glu Phe Gly Lys Met Cys Ser Glu Phe355 360 365 Trp Leu Pro Asp Thr Phe Gly Tyr Ser Ala Gln Leu Pro Gln IleMet 370 375 380 His Gly Cys Gly Ile Arg Arg Phe Leu Thr Gln Lys Leu SerTrp Asn 385 390 395 400 Leu Val Asn Ser Phe Pro His His Thr Phe Phe TrpGlu Gly Leu Asp 405 410 415 Gly Ser Arg Val Leu Val His Phe Pro Pro GlyAsp Ser Tyr Gly Met 420 425 430 Gln Gly Ser Val Glu Glu Val Leu Lys ThrVal Ala Asn Asn Arg Asp 435 440 445 Lys Gly Arg Ala Asn His Ser Ala PheLeu Phe Gly Phe Gly Asp Gly 450 455 460 Gly Gly Gly Pro Thr Gln Thr MetLeu Asp Arg Leu Lys Arg Leu Ser 465 470 475 480 Asn Thr Asp Gly Leu ProArg Val Gln Leu Ser Ser Pro Arg Gln Leu 485 490 495 Phe Ser Ala Leu GluSer Asp Ser Glu Gln Leu Cys Thr Trp Val Gly 500 505 510 Glu Leu Phe LeuGlu Leu His Asn Gly Thr Tyr Thr Thr His Ala Gln 515 520 525 Ile Lys LysGly Asn Arg Glu Cys Glu Arg Ile Leu His Asp Val Glu 530 535 540 Leu LeuSer Ser Leu Ala Leu Ala Arg Ser Ala Gln Phe Leu Tyr Pro 545 550 555 560Ala Ala Gln Leu Gln His Leu Trp Arg Leu Leu Leu Leu Asn Gln Phe 565 570575 His Asp Val Val Thr Gly Ser Cys Ile Gln Met Val Ala Glu Glu Ala 580585 590 Met Cys His Tyr Glu Asp Ile Arg Ser His Gly Asn Thr Leu Leu Ser595 600 605 Ala Ala Ala Ala Ala Leu Cys Ala Gly Glu Pro Gly Pro Glu GlyLeu 610 615 620 Leu Ile Val Asn Thr Leu Pro Trp Lys Arg Ile Glu Val MetAla Leu 625 630 635 640 Pro Lys Pro Gly Gly Ala His Ser Leu Ala Leu ValThr Val Pro Ser 645 650 655 Met Gly Tyr Ala Pro Val Pro Pro Pro Thr SerLeu Gln Pro Leu Leu 660 665 670 Pro Gln Gln Pro Val Phe Val Val Gln GluThr Asp Gly Ser Val Thr 675 680 685 Leu Asp Asn Gly Ile Ile Arg Val LysLeu Asp Pro Thr Gly Arg Leu 690 695 700 Thr Ser Leu Val Leu Val Ala SerGly Arg Glu Ala Ile Ala Glu Gly 705 710 715 720 Ala Val Gly Asn Gln PheVal Leu Phe Asp Asp Val Pro Leu Tyr Trp 725 730 735 Asp Ala Trp Asp ValMet Asp Tyr His Leu Glu Thr Arg Lys Pro Val 740 745 750 Leu Gly Gln AlaGly Thr Leu Ala Val Gly Thr Glu Gly Gly Leu Arg 755 760 765 Gly Ser AlaTrp Phe Leu Leu Gln Ile Ser Pro Asn Ser Arg Leu Ser 770 775 780 Gln GluVal Val Leu Asp Val Gly Cys Pro Tyr Val Arg Phe His Thr 785 790 795 800Glu Val His Trp His Glu Ala His Lys Phe Leu Lys Val Glu Phe Pro 805 810815 Ala Arg Val Arg Ser Ser Gln Ala Thr Tyr Glu Ile Gln Phe Gly His 820825 830 Leu Gln Arg Pro Thr His Tyr Asn Thr Ser Trp Asp Trp Ala Arg Phe835 840 845 Glu Val Trp Ala His Arg Trp Met Asp Leu Ser Glu His Gly PheGly 850 855 860 Leu Ala Leu Leu Asn Asp Cys Lys Tyr Gly Ala Ser Val ArgGly Ser 865 870 875 880 Ile Leu Ser Leu Ser Leu Leu Arg Ala Pro Lys AlaPro Asp Ala Thr 885 890 895 Ala Asp Thr Gly Arg His Glu Phe Thr Tyr AlaLeu Met Pro His Lys 900 905 910 Gly Ser Phe Gln Asp Ala Gly Val Ile GlnAla Ala Tyr Ser Leu Asn 915 920 925 Phe Pro Leu Leu Ala Leu Pro Ala ProSer Pro Ala Pro Ala Thr Ser 930 935 940 Trp Ser Ala Phe Ser Val Ser SerPro Ala Val Val Leu Glu Thr Val 945 950 955 960 Lys Gln Ala Glu Ser SerPro Gln Arg Arg Ser Leu Val Leu Arg Leu 965 970 975 Tyr Glu Ala His GlySer His Val Asp Cys Trp Leu His Leu Ser Leu 980 985 990 Pro Val Gln GluAla Ile Leu Cys Asp Leu Leu Glu Arg Pro Asp Pro 995 1000 1005 Ala GlyHis Leu Thr Ser Gly Gln Pro Pro Glu Ala His Leu Phe Ser 1010 1015 1020Leu Pro Ser Ala Val Pro Val Ala Arg Ala Ser Ala Ser Ala Thr Leu 10251030 1035 1040 Ser Pro Trp Gly Trp Gly Phe Val Cys Arg Arg Leu Trp GlyLeu Leu 1045 1050 1055 Ile Ser Ala Ser Pro Ala 1060 103 26 DNASaccharomyces cerevisiae modified_base (9) a, c, g or t 103 taytggmgngtngarcynga yathaa 26 104 20 DNA Saccharomyces cerevisiae modified_base(6) a, c, g or t 104 gcrtcncccc anckytcrta 20 105 4 PRT ArtificialSequence Description of Artificial Sequence Synthetic peptide 105 HisAsp Glu Leu 1 106 4 PRT Artificial Sequence Description of ArtificialSequence Synthetic peptide 106 Lys Asp Glu Leu 1 107 38 DNA ArtificialSequence Description of Artificial Sequence Synthetic primer 107gcggccgcgg atccccgggt accgagctcg aattcact 38 108 57 DNA ArtificialSequence Description of Artificial Sequence Synthetic primer 108ggggcgcgcc ttaattaacg acctgcaggc atgcaagctt ggcgtaatca tggtcat 57 109 31DNA Artificial Sequence Description of Artificial Sequence Syntheticprimer 109 ttcctcgaga ttcaagcgaa tgagaataat g 31 110 33 DNA ArtificialSequence Description of Artificial Sequence Synthetic primer 110ttgcggccgc gaagttttta aaggaaagag ata 33 111 43 DNA Artificial SequenceDescription of Artificial Sequence Synthetic primer 111 ggcgcgccgagcccgctgac gccaccatcc gtgagaagag ggc 43 112 62 DNA Artificial SequenceDescription of Artificial Sequence Synthetic primer 112 atgtggcggcggccgccacc atgaacacta tccacataat aaaattaccg cttaactacg 60 cc 62 113 45DNA Artificial Sequence Description of Artificial Sequence Syntheticprimer 113 ggcgcgcccc acgcctagca cttttatgga atctacgcta ggtac 45 114 53DNA Artificial Sequence Description of Artificial Sequence Syntheticprimer 114 agtaaaatgc ggccgccacc atgctgctta ccaaaaggtt ttcaaagctg ttc 53115 45 DNA Artificial Sequence Description of Artificial SequenceSynthetic primer 115 ggcgcgcccc gacgtgttct catccatgta tttgtttgta atgac45 116 25 DNA Rattus norvegicus 116 ttcctcactg cagtcttcta taact 25 11726 DNA Rattus norvegicus 117 tggagaccat gaggttccgc atctac 26 118 35 DNARattus norvegicus 118 ttggcgcgcc tccctagtgt accagttgaa ctttg 35 119 32DNA Rattus norvegicus 119 gattaattaa ctcactgcag tcttctataa ct 32

What is claimed is:
 1. A method for producing a human-like glycoproteinin a lower eukaryotic host cell comprising the step of expressing in thecell a mannosidase enzymatic activity that is capable of hydrolyzing anoligosaccharide substrate comprising either or both a Manα1,3 andManα1,6 glycosidic linkage to the extent that at least 10% of theManα1,3 and/or Manα1,6 linkages of the substrate are hydrolyzed in vivo.2. A method for producing a desired N-glycan in a lower eukaryotic hostcell comprising the step of expressing in the cell a mannosidaseenzymatic activity that is capable of hydrolyzing in vivo anoligosaccharide substrate comprising either or both a Manα1,3 andManα1,6 glycosidic linkage wherein the desired N-glycan is producedwithin the host cell at a yield of at least 10 mole percent.
 3. Themethod of claim 2, wherein the desired N-glycan produced is selectedfrom the group consisting of Man₃GlcNAc₂, GlcNAcMan₃GlcNAc₂ andMan₄GlcNAc₂.
 4. The method of claim 2, wherein the desired N-glycan ischaracterized as having at least the oligosaccharide branch Manα1,3(Manα1,6) Manβ1,4-GlcNAc β1,4-GlcNAc-Asn.
 5. The method of claim 1 or 2,wherein the mannosidase enzymatic activity is capable of hydrolyzing invivo both Manα1,3 and Manα1,6 linkages of an oligosaccharide substratecomprising a Manα1,3 and Manα1,6 glycosidic linkage.
 6. The method ofclaim 1 or 2, wherein the oligosaccharide substrate is characterized asManα1,3 (Manα1,6 Manα1,6) Manβ1,4-GlcNAc β1,4-GlcNAc-Asn; Manα1,3(Manα1,3 Manα1,6) Manα1,4-GlcNAc β1,4-GlcNAc-Asn; GlcNAcβ1,2 Manα1,3(Manα1,6 Manα1,6) Manα1,4-GlcNAc β1,4-GlcNAc-Asn; GlcNAcβ1,2 Manα1,3(Manα1,3 Manα1,6) Manα1,4-GlcNAc β1,4-GlcNAc-Asn; Manα1,3 (Manα1,3Manα1,6 Manα1,6) Manβ1,4-GlcNAc β1,4-GlcNAc-Asn; GlcNAcβ1,2 Manα1,3(Manα1,3 Manα1,6 Manα1,6) Manβ1,4-GlcNAc β1,4-GlcNAc-Asn; Manα1,2Manα1,3 (Manα1,3 Manα1,6 Manα1,6) Manα1,4-GlcNAc β1,4-GlcNAc-Asn;Manα1,2 Manα1,3 (Manα1,3 Manα1,6) Manβ1,4-GlcNAc β1,4-GlcNAc-Asn;Manα1,2 Manα1,3 (Manα1,6 Manα1,6) Manβ1,4-GlcNAc β1,4-GlcNAc-Asn or highmannan.
 7. The method of claim 1 or 2, wherein the mannosidase activityis characterized as a Class 2 mannosidase activity.
 8. The method ofclaim 7, wherein the Class 2 mannosidase activity has a substratespecificity for GlcNAcβ1,2 Manα1,3 (Manα1,6 Manα1,6) Manβ1,4-GlcNAcβ1,4-GlcNAc-Asn; GlcNAcβ1,2 Manα1,3 (Manα1,3 Manα1,6) Manβ1,4-GlcNAcβ1,4-GlcNAc-Asn; or GlcNAcβ1,2 Manα1,3 (Manα1,3 Manα1,6 Manα1,6)Manβ1,4-GlcNAc β1,4-GlcNAc-Asn.
 9. The method of claim 7, wherein theClass 2 mannosidase activity is one which is normally found in the Golgiapparatus of a higher eukaryotic host cell.
 10. The method of claim 1 or2, wherein the mannosidase activity is characterized as a Class IIxmannosidase activity.
 11. The method of claim 10, wherein the Class IIxmannosidase activity has a substrate specificity for Manα1,3 (Manα1,6Manα1,6) Manβ1,4-GlcNAc β1,4-GlcNAc-Asn; Manα1,3 (Manα1,3 Manα1,6)Manβ1,4-GlcNAc β1,4-GlcNAc-Asn; or Manα1,2 Manα1,3 (Manα1,3 Manα1,6Manα1,6) Manβ1,4-GlcNAc β1,4-GlcNAc-Asn.
 12. The method of claim 1 or 2,wherein the mannosidase activity is characterized as a Class IIImannosidase activity.
 13. The method of claim 12, wherein the Class IIImannosidase activity has a substrate specificity for (Manα1,6 Manα1,6)Manβ1,4-GlcNAc β1,4-GlcNAc-Asn; (Manβ1,3 Manα1,6) Manβ1,4-GlcNAcβ1,4-GlcNAc-Asn; or high mannans.
 14. The method of claim 1 or 2,wherein the mannosidase activity is overexpressed.
 15. The method ofclaim 1 or 2, wherein the mannosidase is further capable of hydrolyzinga Manα1,2 linkage.
 16. The method of claim 1 or 2, wherein themannosidase activity has a pH optimum of from about 5.0 to about 8.0.17. The method of claim 1 or 2, wherein the mannosidase is furthercapable of hydrolyzing a Manβ1,2 linkage.
 18. The method of claim 1 or2, wherein the mannosidase activity is localized within the secretorypathway of the host cell.
 19. The method of claim 1 or 2, wherein themannosidase activity is expressed from a polypeptide localized within atleast one of the ER, Golgi apparatus or the trans golgi network of thehost cell.
 20. The method of claim 1 or 2, wherein the mannosidaseactivity is expressed from a nucleic acid encoding a polypeptidecomprising a mannosidase catalytic domain fused to a cellular targetingsignal peptide.
 21. The method of claim 20, wherein the mannosidaseactivity is expressed from a niucleic acid comprising sequences thatencode a mannosidase catalytic domain native to the host cell
 22. Themethod of claim 20, wherein the mannosidase activity is expressed from anucleic acid comprising sequences that encode a mannosidase catalyticdomain heterologous to the host cell.
 23. The method of claim 1 or 2,wherein the mannosidase enzymatic activity is selected from the groupconsisting of Arabidopsis thaliana Mannosidase II, C. elegansMannosidase II, Ciona intesiinalis mannosidase II, Drosophilamannosidase II, Human mannosidase II, Mouse mannosidase II, Ratmannosidase II, Human mannosidase IIx, Insect cell mannosidase III,Human lysosomal mannosidase II and Human cytoplasmic mannosidase II. 24.The method of claim 1 or 2, wherein the polypeptide is expressed from anucleic acid comprising sequences that encode a target peptide native tothe host cell.
 25. The method of claim 1 or 2, wherein the polypeptideis expressed from a nucleic acid comprising sequences that encode atarget peptide heterologous to the mannosidase catalytic domain.
 26. Themethod of claim 1 or 2, further comprising the step of isolating theglycoprotein from the host cell.
 27. The method of claim 1 or 2, whereinthe host cell is selected from the group consisting of Pichia pastoris,Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichiamembranaefaciens, Pichia opuntiae, Pichia thermotolerans, Pichiasalictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichiamethanolica, Pichia sp., Saccharomyces cerevisiae, Saccharomyces sp.,Hansenula polymorpha, Kluyveromyces sp., Kluyveromyces lactis, Candidaalbicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae,Trichoderma reesei, Chrysosporium lucknowense, Fusarium sp., Fusariumgramineum, Fusarium venenatum and Neurospora crassa.
 28. The method ofclaim 27, wherein the host cell is Pichia pastoris.
 29. The method ofclaim 1 or 2, wherein the glycoprotein is a therapeutic protein.
 30. Themethod of claim 29, wherein the therapeutic protein is selected from thegroup consisting of erythropoietin, cytokines, coagulation factors,soluble IgE receptor α-chain, IgG, IgG fragments, IgM, interleukins,urokinase, chymase, urea trypsin inhibitor, IGF-binding protein,epidermal growth factor, growth hormone-releasing factor, annexin Vfusion protein, angiostatin, vascular endothelial growth factor-2,myeloid progenitor inhibitory factor-1, osteoprotegerin, α-1-antitrypsinand α-feto protein.
 31. A nucleic acid library comprising at least twodifferent genetic constructs, wherein at least one genetic constructcomprises a nucleic acid fragment encoding a mannosidase class 2, IIx orIII catalytic domain ligated in-frame with a nucleic acid fragmentencoding a cellular targeting signal peptide which it is not normallyassociated with.
 32. The library of claim 31, wherein the mannosidasecatalytic domain is selected from the group consisting of Arabidopsisthaliana Mannosidase II, C. elegans Mannosidase II, Ciona intestinalismannosidase II, Drosophila mannosidase II, Human mannosidase II, Mousemannosidase II, Rat mannosidase II, Human mannosidase IIx, Insect cellmannosidase III, Human lysosomal mannosidase II and Human cytoplasmicmannosidase II.
 33. The library of claim 31, wherein the nucleic acidfragment encoding a cellular targeting peptide is selected from thegroup consisting of: Saccharomyces GLS1, Saccharomyces MNS1,Saccharomyces SEC 12, Pichia SEC, Pichia OCH1, Saccharomyces MNN9,Saccharomyces VAN1, Saccharomyces ANP1, Saccharomyces HOC1,Saccharomyces MNN10, Saccharomyces MNN11, Saccharomyces MNT1, Pichia D2,Pichia D9, Pichia J3, Saccharomyces KTR1, Saccharomyces KTR2,Kluyveromyces GnTI, Saccharomyces MNN2, Saccharomyces MNN5,Saccharomyces YUR1, Saccharomyces MNN1, and Saccharomyces MNN6.
 34. Avector comprising a fusion construct derived from a library of any oneof claims 31-33 operably linked to an expression control sequence,wherein said cellular targeting signal peptide is targeted to at leastone of the ER, Golgi or trans-Golgi network.
 35. The vector of claim 34,wherein the expression control sequence is inducible or constitutive.36. The vector of claim 34 which, upon expression in a host cell,encodes a mannosidase activity involved in producing GlcNAcMan₃GlcNAc₂Man₃GlcNAc₂ or Man₄GlcNAc₂ in vivo.
 37. A host cell comprising at leastone vector of claim
 36. 38. A host cell comprising at least one vectorselected from the group of vectors designated pKD53, pKD1, pKD5, pKD6and pKD16.
 39. A chimeric polypeptide comprising a mannosidase catalyticdomain fused in-frame to a targeting signal peptide and, upon expressionin a lower eukaryotic host cell, capable of hydrolyzing in vivo anoligosaccharide substrate comprising either or both a Manα1,3 andManα1,6 glycosidic linkage to the extent that at least 10% of theManα1,3 and/or Manα1,6 linkages of the substrate are hydrolyzed in vivo.40. A chimeric polypeptide comprising a mannosidase catalytic domainfused in-frame to a targeting signal peptide and, upon expression in alower eukaryotic host cell, capable of hydrolyzing in vivo anoligosaccharide substrate comprising a Manα1,3, Manα1,6, or Manα1,2glycosidic linkage to the extent that a detectable moiety of theManα1,3, Manα1,6 or Manα1,2 linkage of the substrate is hydrolyzed invivo.
 41. A nucleic acid encoding a chimeric polypeptide of claim 39.42. A host cell comprising a chimeric polypeptide of claim
 39. 43. Ahost cell comprising a nucleic acid of claim
 41. 44. A glycoproteinproduced in a host cell of claim 42 or claim
 43. 45. An N-glycanproduced in a host cell of claim 42 or claim
 43. 46. The N-glycan ofclaim 45, wherein the N-glycan is characterized as uniform.
 47. Aglycoprotein produced by the method of claim 1 or claim
 2. 48. AnN-glycan produced by the method of claim 1 or claim
 2. 49. The N-glycanof claim 48, wherein the N-glycan is characterized as uniform.
 50. Anisolated polynucleotide comprising or consisting of a nucleic acidsequence selected from the group consisting of: (a) SEQ ID NO: 92(C.elegans FROM FIG. 23); (b) at least about 90% similar to the aminoacid residues of the donor nucleotide binding site of SEQ ID NO: 92; (c)a nucleic acid sequence at least 92%, at least 95%, at least 98%, atleast 99% or at least 99.9% identical to SEQ ID NO: 93; (d) a nucleicacid sequence that encodes a conserved polypeptide having the amino acidsequence of SEQ ID NO: 92; (e) a nucleic acid sequence that encodes apolypeptide at least 78%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 98%, at least 99% or at least 99.9% identical to SEQID NO:92; (f) a nucleic acid sequence that hybridizes under stringentconditions to SEQ ID NO:92; and (g) a nucleic acid sequence comprising afragment of any one of (a)-(f) that is at least 60 contiguousnucleotides in length.
 51. An isolated polynucleotide comprising orconsisting of a nucleic acid sequence selected from the group consistingof: (a) SEQ ID NO: 93(rat FROM FIG. 23); (b) at least about 95% similarto the amino acid residues of the donor nucleotide binding site of SEQID NO: 93; (c) a nucleic acid sequence at least 95%, at least 98%, atleast 99% or at least 99.9% identical to SEQ ID NO: 93; (d) a nucleicacid sequence that encodes a conserved polypeptide having the amino acidsequence of SEQ ID NO: 93; (e) a nucleic acid sequence that encodes apolypeptide at least 97%, at least 98%, at least 99% or at least 99.9%identical to SEQ ID NO: 93; (f) a nucleic acid sequence that hybridizesunder stringent conditions to SEQ ID NO: 93; and (g) a nucleic acidsequence comprising a fragment of any one of (a)-(f) that is at least 60contiguous nucleotides in length.
 52. An isolated polynucleotidecomprising or consisting of a nucleic acid sequence selected from thegroup consisting of: (a) SEQ ID NO: 94(Ciona FROM FIG. 23); (b) at leastabout 90% similar to the amino acid residues of the donor nucleotidebinding site of SEQ ID NO: 94; (c) a nucleic acid sequence at least 92%,at least 95%, at least 98%, at least 99% or at least 99.9% identical toSEQ ID NO: 94; (d) a nucleic acid sequence that encodes a conservedpolypeptide having the amino acid sequence of SEQ ID NO: 94; (e) anucleic acid sequence that encodes a polypeptide at least 73%, at least80%, at least 85%, at least 90%, at least 95%, at least 98%, at least99% or at least 99.9% identical to SEQ ID NO: 94; (f) a nucleic acidsequence that hybridizes under stringent conditions to SEQ ID NO: 94;and (g) a nucleic acid sequence comprising a fragment of any one of(a)-(f) that is at least 60 contiguous nucleotides in length.
 53. Anisolated polynucleotide comprising or consisting of a nucleic acidsequence selected from the group consisting of: (a) SEQ ID NO:95(Arabidopsis FROM FIG. 23); (b) at least about 95% similar to theamino acid residues of the donor nucleotide binding site of SEQ ID NO:95; (c) a nucleic acid sequence at least 96%, at least 98%, at least 99%or at least 99.9% identical to SEQ ID NO: 95; (d) a nucleic acidsequence that encodes a conserved polypeptide having the amino acidsequence of SEQ ID NO: 95; (e) a nucleic acid sequence that encodes apolypeptide at least 95%, at least 98%, at least 99% or at least 99.9%identical to SEQ ID NO: 95; (f) a nucleic acid sequence that hybridizesunder stringent conditions to SEQ ID NO: 95; and (g) a nucleic acidsequence comprising a fragment of any one of (a)-(f) that is at least 60contiguous nucleotides in length.
 54. A modified polynucleotidecomprising or consisting of a nucleic acid sequence selected from thegroup consisting of the conserved regions SEQ ID NO: 5-SEQ ID NO: 15wherein the encoded polypeptide is involved in hydrolyzing a Manα1,3and/or a Manα1,6 glycosidic linkage of an oligosaccharide.
 55. Amodified polynucleotide comprising or consisting of a nucleic acidsequence selected from the group consisting of the conserved regions ofSEQ ID NO: 49-SEQ ID NO: 59 wherein the encoded polypeptide is involvedin hydrolyzing a Manα1,3 and/or a Manα1,6 glycosidic linkage of anoligosaccharide.
 56. A vector selected from the group consisting ofpKD53, pKD1, pKD5, pKD6 and pKD16.